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The sympathetic nervous system (SNS) consists largely of two different types of components: neurons that release the neurotransmitter norepinephrine (NE, noradrenaline) to modulate homeostasis of the innevrvated effector organ or tissue and adrenal chromaffin cells, which synthesize and secrete the hormone epinephrine (Epi, adrenaline) and some NE into the blood circulation to act at distant organs and tissues that are not directly innervated by the SNS. Like almost every physiological process in the human body, G protein-coupled receptors (GPCRs) tightly modulate both NE release from sympathetic neuronal terminals and catecholamine (CA) secretion from the adrenal medulla. Regulator of G protein Signaling (RGS) proteins, acting as guanosine triphosphatase (GTPase)-activating proteins (GAPs) for the Gα subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins), play a central role in silencing G protein signaling from a plethora of GPCRs. Certain RGS proteins and, in particular, RGS4, have been implicated in regulation of SNS activity and of adrenal chromaffin cell CA secretion. More specifically, recent studies have implicated RGS4 in regulation of NE release from cardiac sympathetic neurons by means of terminating free fatty acid receptor (FFAR)-3 calcium signaling and in regulation of NE and Epi secretion from the adrenal medulla by means of terminating cholinergic calcium signaling in adrenal chromaffin cells. Thus, in this review, we provide an overview of the current literature on the involvement of RGS proteins, with a particular focus on RGS4, in these two processes, i.e., NE release from sympathetic nerve terminals & CA secretion from adrenal chromaffin cells. We also highlight the therapeutic potential of RGS4 pharmacological manipulation for diseases characterized by sympathetic dysfunction or SNS hyperactivity, such as heart failure and hypertension., (© 2024 The Author(s). Published by IMR Press.)

High voltage-gated Ca 2+ channels (HVCCs) shape the electrical activity and control hormone release in most endocrine cells. HVCCs are multi-subunit protein complexes formed by the pore-forming α 1 and the auxiliary β, α 2 δ and γ subunits. Four genes code for the α 2 δ isoforms. At the mRNA level, mouse chromaffin cells (MCCs) express predominantly the CACNA2D1 gene coding for the α 2 δ-1 isoform. Here we show that α 2 δ-1 deletion led to ∼60% reduced HVCC Ca 2+ influx with slower inactivation kinetics. Pharmacological dissection showed that HVCC composition remained similar in α 2 δ-1 -/- MCCs compared to wild-type (WT), demonstrating that α 2 δ-1 exerts similar functional effects on all HVCC isoforms. Consistent with reduced HVCC Ca 2+ influx, α 2 δ-1 -/- MCCs showed reduced spontaneous electrical activity with action potentials (APs) having a shorter half-maximal duration caused by faster rising and decay slopes. However, the induced electrical activity showed opposite effects with α 2 δ-1 -/- MCCs displaying significantly higher AP frequency in the tonic firing mode as well as an increase in the number of cells firing AP bursts compared to WT. This gain-of-function phenotype was caused by reduced functional activation of Ca 2+ -dependent K + currents. Additionally, despite the reduced HVCC Ca 2+ influx, the intracellular Ca 2+ transients and vesicle exocytosis or endocytosis were unaltered in α 2 δ-1 -/- MCCs compared to WT during sustained stimulation. In conclusion, our study shows that α 2 δ-1 genetic deletion reduces Ca 2+ influx in cultured MCCs but leads to a paradoxical increase in catecholamine secretion due to increased excitability. KEY POINTS: Deletion of the α 2 δ-1 high voltage-gated Ca 2+ channel (HVCC) subunit reduces mouse chromaffin cell (MCC) Ca 2+ influx by ∼60% but causes a paradoxical increase in induced excitability. MCC intracellular Ca 2+ transients are unaffected by the reduced HVCC Ca 2+ influx. Deletion of α 2 δ-1 reduces the immediately releasable pool vesicle exocytosis but has no effect on catecholamine (CA) release in response to sustained stimuli. The increased electrical activity and CA release from MCCs might contribute to the previously reported cardiovascular phenotype of patients carrying α 2 δ-1 loss-of-function mutations., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

MicroGraphited-Diamond-Multi Electrode Arrays (μG-D-MEAs) can be successfully used to reveal, in real time, quantal exocytotic events occurring from many individual neurosecretory cells and/or from many neurons within a network. As μG-D-MEAs arrays are patterned with up to 16 sensing microelectrodes, each of them recording large amounts of data revealing the exocytotic activity, the aim of this work was to support an adequate analysis code to speed up the signal detection. The cutting-edge technology of microGraphited-Diamond-Multi Electrode Arrays (μG-D-MEAs) has been implemented with an automated analysis code (APE, Amperometric Peak Analysis) developed using Matlab R2022a software to provide easy and accurate detection of amperometric spike parameters, including the analysis of the pre-spike foot that sometimes precedes the complete fusion pore dilatation. Data have been acquired from cultured PC12 cells, either collecting events during spontaneous exocytosis or after L-DOPA incubation. Validation of the APE code was performed by comparing the acquired spike parameters with those obtained using Quanta Analysis (Igor macro) by Mosharov et al.

In studies exploring the potential for nanosecond duration electric pulses to serve as a novel modality for neuromodulation, we found that a 5 ns pulse triggers an immediate rise in [Ca2+]i in isolated bovine adrenal chromaffin cells. To facilitate ongoing efforts to understand underlying mechanisms and to work toward carrying out investigations in cells in situ, we describe the suitability and advantages of using isolated murine adrenal chromaffin cells expressing, in a Cre-dependent manner, the genetically-encoded Ca2+indicator GCaMP6f. Initial experiments confirmed that Ca2+ responses evoked by a 5 ns pulse were similar between fluorescent Ca2+ indicator-loaded murine and bovine chromaffin cells, thereby establishing that 5 ns-elicited excitation of chromaffin cells occurs reproducibly across species. In GCaMP6f-expressing murine chromaffin cells, spontaneous Ca2+ activity as well as nicotinic receptor agonist- and 5 ns evoked-Ca2+ responses consistently displayed similar kinetic characteristics as those in dye-loaded cells but with two-twentyfold greater amplitudes and without photobleaching. The high signal-to-noise ratio of evoked Ca2+ responses as well as spontaneous Ca2+ activity was observed in cells derived from Sox10-Cre, conditional GCaMP6f mice or TH-Cre, conditional GCaMP6f mice, although the number of cells expressing GCaMP6f at sufficiently high levels for achieving high signal-to-noise ratios was greater in Sox10-Cre mice. As in bovine cells, Ca2+ responses elicited in murine GCaMP6f-expressing cells by a 5 ns pulse were mediated by the activation of voltage-gated Ca2+ channels but not tetrodotoxin-sensitive voltage-gated Na+ channels. We conclude that genetically targeting GCaMP6f expression to murine chromaffin cells represents a sensitive and valuable approach to investigate spontaneous, receptor agonist- and nanosecond electric pulse-induced Ca2+ responses in vitro. This approach will also facilitate future studies investigating the effects of ultrashort electric pulses on cells in ex vivo slices of adrenal gland, which will lay the foundation for using nanosecond electric pulses to stimulate neurosecretion in vivo., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Viola et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Hofmeister effects have often been ignored in living organisms, although they affect the activity and functions of biological molecules. Herein, amperometry has been applied to show that the vesicular content, dynamics of exocytosis and vesicles opening, depend on the anionic species treatment. Compared to 100 μM Cl - treated chromaffin cells, a similar number of catecholamine molecules is released after chaotropic anions (ClO 4 - and SCN - ) treatment, even though the vesicular catecholamine content significantly increases, suggesting a lower release fraction. In addition, there are opposite effects on the dynamics of vesicles release (shorter duration) and vesicle opening (longer duration) for chaotropic anions treated cells. Our results show anion-dependent vesicle release, vesicle opening, and vesicular content, providing understanding of the pharmacological and pathological processes induced by inorganic ions.

The hypersecretory phenotype of adrenal chromaffin cells (CCs) from early spontaneously hypertensive rats (SHRs) mainly results from enhanced Ca 2+ -induced Ca 2+ -release (CICR). A key question is if these abnormalities can be traced to the prehypertensive stage. Spontaneous and stimulus-induced catecholamine exocytosis, intracellular Ca 2+ signals, and dense-core granule size and density were examined in CCs from prehypertensive and hypertensive SHRs and compared with age-matched Wistar-Kyoto rats (WKY). During the prehypertensive stage, the depolarization-elicited catecholamine exocytosis was ~ 2.9-fold greater in SHR than in WKY CCs. Interestingly, in half of CCs the exocytosis was indistinguishable from WKY CCs, while it was between 3- and sixfold larger in the other half. Likewise, caffeine-induced exocytosis was ~ twofold larger in prehypertensive SHR. Accordingly, depolarization and caffeine application elicited [Ca 2+ ]i rises ~ 1.5-fold larger in prehypertensive SHR than in WKY CCs. Ryanodine reduced the depolarization-induced secretion in prehypertensive SHR by 57%, compared to 14% in WKY CCs, suggesting a greater contribution of intracellular Ca 2+ release to exocytosis. In SHR CCs, the mean spike amplitude and charge per spike were significantly larger than in WKY CCs, regardless of age and stimulus type. This difference in granule content could explain in part the enhanced exocytosis in SHR CCs. However, electron microscopy did not reveal significant differences in granule size between SHRs and WKY rats' adrenal medulla. Nonetheless, preSHR and hypSHR display 63% and 82% more granules than WKY, which could explain in part the enhanced catecholamine secretion. The mechanism responsible for the heterogeneous population of prehypertensive SHR CCs and the bias towards secreting more medium and large granules remains unexplained., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Transformation of flat membrane into round vesicles is generally thought to underlie endocytosis and produce speed-, amount-, and vesicle-size-specific endocytic modes. Visualizing depolarization-induced exocytic and endocytic membrane transformation in live neuroendocrine chromaffin cells, we found that flat membrane is transformed into Λ-shaped, Ω-shaped, and O-shaped vesicles via invagination, Λ-base constriction, and Ω-pore constriction, respectively. Surprisingly, endocytic vesicle formation is predominantly from not flat-membrane-to-round-vesicle transformation but calcium-triggered and dynamin-mediated closure of (1) Ω profiles formed before depolarization and (2) fusion pores (called kiss-and-run). Varying calcium influxes control the speed, number, and vesicle size of these pore closures, resulting in speed-specific slow (more than ∼6 s), fast (less than ∼6 s), or ultrafast (<0.6 s) endocytosis, amount-specific compensatory endocytosis (endocytosis = exocytosis) or overshoot endocytosis (endocytosis > exocytosis), and size-specific bulk endocytosis. These findings reveal major membrane transformation mechanisms underlying endocytosis, diverse endocytic modes, and exocytosis-endocytosis coupling, calling for correction of the half-a-century concept that the flat-to-round transformation predominantly mediates endocytosis after physiological stimulation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

γ-Aminobutyric acid (GABA) is thought to play a paracrine role in adrenal medullary chromaffin (AMC) cells. Comparative physiological and immunocytochemical approaches were used to address the issue of how the paracrine function of GABA in AMC cells is established. GABA A receptor Cl - channel activities in AMC cells of rats and mice, where corticosterone is the major glucocorticoid, were much smaller than those in AMC cells of guinea-pigs and cattle, where cortisol is the major. The extent of enhancement of GABA A receptor α3 subunit expression in rat pheochromocytoma (PC12) cells by cortisol was larger than that by corticosterone in parallel with their glucocorticoid activities. Thus, the species difference in GABA A receptor expression may be ascribed to a difference in glucocorticoid activity between corticosterone and cortisol. GABA A receptor Cl - channel activity in mouse AMC cells was enhanced by allopregnanolone, as noted with that in guinea-pig AMC cells, and the enzymes involved in allopregnanolone production were immunohistochemically detected in the zona fasciculata in both mice and guinea pigs. The expression of glutamic acid decarboxylase 67 (GAD67), one of the GABA synthesizing enzymes, increased after birth, whereas GABA A receptors already developed at birth. Stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, but not nicotinic or muscarinic receptors, in PC12 cells, resulted in an increase in GAD67 expression in a protein-kinase A-dependent manner. The results indicate that glucocorticoid and PACAP are mainly responsible for the expressions of GABA A receptors and GAD67 involved in GABA signaling in AMC cells, respectively., (© 2021 International Society for Neurochemistry.)

Synaptophysin (syp) is a major integral membrane protein of secretory vesicles. Previous work has demonstrated functions for syp in synaptic vesicle cycling, endocytosis, and synaptic plasticity, but the role of syp in the process of membrane fusion during Ca 2+ -triggered exocytosis remains poorly understood. Furthermore, although syp resides on both large dense-core and small synaptic vesicles, its role in dense-core vesicle function has received less attention compared with synaptic vesicle function. To explore the role of syp in membrane fusion and dense-core vesicle function, we used amperometry to measure catecholamine release from single vesicles in male and female mouse chromaffin cells with altered levels of syp and the related tetraspanner protein synaptogyrin (syg). Knocking out syp slightly reduced the frequency of vesicle fusion events below wild-type (WT) levels, but knocking out both syp and syg reduced the frequency 2-fold. Knocking out both proteins stabilized initial fusion pores, promoted fusion pore closure (kiss-and-run), and reduced late-stage fusion pore expansion. Introduction of a syp construct lacking its C-terminal dynamin-binding domain in syp knock-outs (KOs) increased the duration and fraction of kiss-and-run events, increased total catecholamine release per event, and reduced late-stage fusion pore expansion. These results demonstrated that syp and syg regulate dense-core vesicle function at multiple stages to initiate fusion, control the choice of mode between full-fusion and kiss-and-run, and influence the dynamics of both initial and late-stage fusion pores. The transmembrane domain (TMD) influences small initial fusion pores, and the C-terminal domain influences large late-stage fusion pores, possibly through an interaction with dynamin. SIGNIFICANCE STATEMENT The secretory vesicle protein synaptophysin (syp) is known to function in synaptic vesicle cycling, but its roles in dense-core vesicle functions, and in controlling membrane fusion during Ca 2+ -triggered exocytosis remain unclear. The present study used amperometry recording of catecholamine release from endocrine cells to assess the impact of syp and related proteins on membrane fusion. A detailed analysis of amperometric spikes arising from the exocytosis of single vesicles showed that these proteins influence fusion pores at multiple stages and control the choice between kiss-and-run and full-fusion. Experiments with a syp construct lacking its C terminus indicated that the transmembrane domain (TMD) influences the initial fusion pore, while the C-terminal domain influences later stages after fusion pore expansion., (Copyright © 2021 the authors.)

Reduced expression of the plasma membrane citrate transporter INDY (acronym I'm Not Dead, Yet) extends life span in lower organisms. Deletion of the mammalian Indy (mIndy) gene in rodents improves metabolism via mechanisms akin to caloric restriction, known to lower blood pressure (BP) by sympathoadrenal inhibition. We hypothesized that mIndy deletion attenuates sympathoadrenal support of BP. Continuous arterial BP and heart rate (HR) were reduced in mINDY-KO mice. Concomitantly, urinary catecholamine content was lower, and the decreases in BP and HR by mIndy deletion were attenuated after autonomic ganglionic blockade. Catecholamine biosynthesis pathways were reduced in mINDY-KO adrenals using unbiased microarray analysis. Citrate, the main mINDY substrate, increased catecholamine content in pheochromocytoma cells, while pharmacological inhibition of citrate uptake blunted the effect. Our data suggest that deletion of mIndy reduces sympathoadrenal support of BP and HR by attenuating catecholamine biosynthesis. Deletion of mIndy recapitulates beneficial cardiovascular and metabolic responses to caloric restriction, making it an attractive therapeutic target.

The present work, using chromaffin cells in rat adrenal slices (RCCs), aims to describe what type of ionic current alterations induced by zinc underlies their effects reported on synaptic transmission. Thus, Zn 2+ blocked calcium channels of RCCs in a time- and concentration-dependent manner with an IC 50 of 391 μM. This blockade was partially reversed upon washout and was greater at more depolarizing holding potentials (i.e. 32 ± 5% at -110 mV, and 43 ± 6% at -50 mV, after 5 min perfusion). In ω-toxins-sensitive calcium channels (N-, P- and Q-types), Zn 2+ caused a lower blockade of I Ca , 33.3%, than in ω-toxins-resistant ones (L-type, 55.3%; and R-type, 90%). This compound inhibited calcium current at all test potentials and shows a shift of the I-V curve to more depolarized values of about 10 mV. The sodium current was not blocked by acute application of high Zn 2+ concentrations. Voltage-dependent potassium current was marginally affected by high Zn 2+ concentrations showing no concentration-dependence. Nevertheless, calcium- and voltage-dependent potassium current was drastically depressed in a dose-dependent manner, with an IC 50 of 453 μM. This blockade was related to the prevention of Ca 2+ influx through voltage-dependent calcium channels coupled to BK channels. Under current-clamp conditions, RCCs exhibit a resting potential of -50.7 mV, firing spontaneous APs (1-2 spikes/s) generated by the opening of Na + and Ca 2+ -channels, and terminated by the activation of voltage and Ca 2+ -activated K + -channels (BK). We found that the blockade of these ionic currents by Zn 2+ led to a drastic alteration of cellular excitability with a depolarization of the membrane potential, the slowdown and broadening of the APs and the severe reduction of the after hyperpolarization (AHP) which led to a decrease in the APs firing frequency. Taken together, these results point to a neurotoxic action evoked by zinc that is associated with changes to cellular excitability by blocking the ionic currents responsible for both the neurotransmitter release and the action potentials firing., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Current models emphasize that membrane voltage (Vm) depolarization-induced Ca 2+ influx triggers the fusion of vesicles to the plasma membrane. In sympathetic adrenal chromaffin cells, activation of a variety of G protein coupled receptors (GPCRs) can inhibit quantal size (QS) through the direct interaction of G protein Giβγ subunits with exocytosis fusion proteins. Here we report that, independently from Ca 2+ , Vm (action potential) per se regulates the amount of catecholamine released from each vesicle, the QS. The Vm regulation of QS was through ATP-activated GPCR-P2Y 12 receptors. D76 and D127 in P2Y 12 were the voltage-sensing sites. Finally, we revealed the relevance of the Vm dependence of QS for tuning autoinhibition and target cell functions. Together, membrane voltage per se increases the quantal size of dense-core vesicle release of catecholamine via Vm → P2Y 12 (D76/D127) → Giβγ → QS → myocyte contractility, offering a universal Vm-GPCR signaling pathway for its functions in the nervous system and other systems containing GPCRs., Competing Interests: The authors declare no competing interest.

Objective: Phosphoinositides play a regulatory role in clathrin-mediated endocytosis. However, their involvement in clathrin-independent endocytosis termed rapid endocytosis (RE), which is the mode of vesicle recycling during neurotransmitter release by transient fusion (known as kiss-and-run), has not been investigated. Here, we used patch-clamp recording of whole-cell membrane capacitance in adrenal chromaffin cells (ACC) to monitor changes of RE kinetics in response to pharmacological alteration of phosphatidylinositol-4,5-biphosphate (PI(4,5)P 2 ) level by phenylarsine oxide (PAO) or antibody against phosphatidylinositol 4-kinase (Ab PI4K )., Results: We found that PAO and Ab PI4K significantly abrogated RE kinetics. Infusion of PI(4,5)P 2 through the patch pipette potentiated RE kinetics and reversed PAO- and Ab PI4K -induced blockade of RE. Similarly, the application of the bifunctional thiol dithiothreitol (DTT) to PAO-treated cells completely prevented the inhibitory effect of PAO on RE. These findings indicate that PI(4,5)P 2 is implicated in the signaling (mechanistic) process of RE in ACC., Competing Interests: The author declares that he has no competing interests., (Copyright © 2020 Fouad Azizi.)

We previously reported that a single 5 ns high intensity electric pulse (NEP) caused an E-field-dependent decrease in peak inward voltage-gated Na+ current (INa) in isolated bovine adrenal chromaffin cells. This study explored the effects of a pair of 5 ns pulses on INa recorded in the same cell type, and how varying the E-field amplitude and interval between the pulses altered its response. Regardless of the E-field strength (5 to 10 MV/m), twin NEPs having interpulse intervals ≥ than 5 s caused the inhibition of TTX-sensitive INa to approximately double relative to that produced by a single pulse. However, reducing the interval from 1 s to 10 ms between twin NEPs at E-fields of 5 and 8 MV/m but not 10 MV/m decreased the magnitude of the additive inhibitory effect by the second pulse in a pair on INa. The enhanced inhibitory effects of twin vs single NEPs on INa were not due to a shift in the voltage-dependence of steady-state activation and inactivation but were associated with a reduction in maximal Na+ conductance. Paradoxically, reducing the interval between twin NEPs at 5 or 8 MV/m but not 10 MV/m led to a progressive interval-dependent recovery of INa, which after 9 min exceeded the level of INa reached following the application of a single NEP. Disrupting lipid rafts by depleting membrane cholesterol with methyl-β-cyclodextrin enhanced the inhibitory effects of twin NEPs on INa and ablated the progressive recovery of this current at short twin pulse intervals, suggesting a complete dissociation of the inhibitory effects of twin NEPs on this current from their ability to stimulate its recovery. Our results suggest that in contrast to a single NEP, twin NEPs may influence membrane lipid rafts in a manner that enhances the trafficking of newly synthesized and/or recycling of endocytosed voltage-gated Na+ channels, thereby pointing to novel means to regulate ion channels in excitable cells., Competing Interests: The authors have declared that no competing interests exist.

Aim: It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by Ca 2+ entry through voltage-dependent Ca 2+ channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca 2+ current and intracellular Ca 2+ increase. In this work we investigated if such a mechanism contributes to secretory vesicle exocytosis in mouse chromaffin cells., Methods: Exocytosis was evaluated by patch-clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic Ca 2+ was estimated using epifluorescence microscopy and fluo-8 (salt form)., Results: Cells stimulated by brief depolatizations in absence of extracellular Ca +2 show moderate but consistent exocytosis, even in presence of high cytosolic BAPTA concentration and pharmacological inhibition of Ca +2 release from intracellular stores. This exocytosis is tightly dependent on membrane potential, is inhibited by neurotoxin Bont-B (cleaves the v-SNARE synaptobrevin), is very fast (saturates with time constant <10 ms), it is followed by a fast endocytosis sensitive to the application of an anti-dynamin monoclonal antibody, and recovers after depletion in <5 s. Finally, this exocytosis was inhibited by: (i) ω-agatoxin IVA (blocks P/Q-type Ca 2+ channel gating), (ii) in cells from knock-out P/Q-type Ca 2+ channel mice, and (iii) transfection of free synprint peptide (interferes in P/Q channel-exocytic proteins association)., Conclusion: We demonstrated that Ca 2+ -independent and voltage-dependent exocytosis is present in chromaffin cells. This process is tightly coupled to membrane depolarization, and is able to support secretion during action potentials at low basal rates. P/Q-type Ca 2+ channels can operate as voltage sensors of this process., (© 2019 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Synaptic disruption and altered neurotransmitter release occurs in the brains of patients and in murine models of neurodegenerative diseases (NDDs). During the last few years, evidence has accumulated suggesting that the sympathoadrenal axis is also affected as disease progresses. Here, we review a few studies done in adrenal medullary chromaffin cells (CCs), that are considered as the amplifying arm of the sympathetic nervous system; the sudden fast exocytotic release of their catecholamines-stored in noradrenergic and adrenergic cells-plays a fundamental role in the stress fight-or-flight response. Bulk exocytosis and the fine kinetics of single-vesicle exocytotic events have been studied in mouse models carrying a mutation linked to NDDs. For instance, in R6/1 mouse models of Huntington's disease (HD), mutated huntingtin is overexpressed in CCs; this causes decreased quantal secretion, smaller quantal size and faster kinetics of the exocytotic fusion pore, pore expansion, and closure. This was accompanied by decreased sodium current, decreased acetylcholine-evoked action potentials, and attenuated [Ca 2+ ]c transients with faster Ca 2+ clearance. In the SOD1 G93A mouse model of amyotrophic lateral sclerosis (ALS), CCs exhibited secretory single-vesicle spikes with a slower release rate but higher exocytosis. Finally, in the APP/PS1 mouse model of Alzheimer's disease (AD), the stabilization, expansion, and closure of the fusion pore was faster, but the secretion was attenuated. Additionally, α-synuclein that is associated with Parkinson's disease (PD) decreases exocytosis and promotes fusion pore dilation in adrenal CCs. Furthermore, Huntington-associated protein 1 (HAP1) interacts with the huntingtin that, when mutated, causes Huntington's disease (HD); HAP1 reduces full fusion exocytosis by affecting vesicle docking and controlling fusion pore stabilization. The alterations described here are consistent with the hypothesis that central alterations undergone in various NDDs are also manifested at the peripheral sympathoadrenal axis to impair the stress fight-or-flight response in patients suffering from those diseases. Such alterations may occur: (i) primarily by the expression of mutated disease proteins in CCs; (ii) secondarily to stress adaptation imposed by disease progression and the limitations of patient autonomy.

Phosphatidylserine (PS), a negatively charged phospholipid present predominantly at the inner leaflet of the plasma membrane, has been widely implicated in many cellular processes including membrane trafficking. Along this line, PS has been demonstrated to be important for endocytosis, however, the involved mechanisms remain uncertain. By monitoring clathrin-mediated endocytosis (CME) of single vesicles in mouse chromaffin cells using cell-attached capacitance measurements that offer millisecond time resolution, we demonstrate in the present study that the fission-pore duration is reduced by PS addition, indicating a stimulatory role of PS in regulating the dynamics of vesicle fission during CME. Furthermore, our results show that the PS-mediated effect on the fission-pore duration is Ca 2+ -dependent and abolished in the absence of synaptotagmin 1 (Syt1), implying that Syt1 is necessary for the stimulatory role of PS in vesicle fission during CME. Consistently, a Syt1 mutant with a defective PS-Syt1 interaction increases the fission-pore duration. Taken together, our study suggests that PS-Syt1 interaction may be critical in regulating fission dynamics during CME., (© 2019 International Society for Neurochemistry.)

Chromaffin cells (CCs) of the adrenal gland and the sympathetic nervous system produce the catecholamines (epinephrine and norepinephrine; EPI and NE) needed to coordinate the bodily "fight-or-flight" response to fear, stress, exercise, or conflict. EPI and NE release from CCs is regulated both neurogenically by splanchnic nerve fibers and nonneurogenically by hormones (histamine, corticosteroids, angiotensin, and others) and paracrine messengers [EPI, NE, adenosine triphosphate, opioids, γ-aminobutyric acid (GABA), etc.]. The "stimulus-secretion" coupling of CCs is a Ca 2+ -dependent process regulated by Ca 2+ entry through voltage-gated Ca 2+ channels, Ca 2+ pumps, and exchangers and intracellular organelles (RE and mitochondria) and diffusible buffers that provide both Ca 2+ -homeostasis and Ca 2+ -signaling that ultimately trigger exocytosis. CCs also express Na + and K + channels and ionotropic (nAChR and GABA A ) and metabotropic receptors (mACh, PACAP, β-AR, 5-HT, histamine, angiotensin, and others) that make CCs excitable and responsive to autocrine and paracrine stimuli. To maintain high rates of E/NE secretion during stressful conditions, CCs possess a large number of secretory chromaffin granules (CGs) and members of the soluble NSF-attachment receptor complex protein family that allow docking, fusion, and exocytosis of CGs at the cell membrane, and their recycling. This article attempts to provide an updated account of well-established features of the molecular processes regulating CC function, and a survey of the as-yet-unsolved but important questions relating to CC function and dysfunction that have been the subject of intense research over the past 15 years. Examples of CCs as a model system to understand the molecular mechanisms associated with neurodegenerative diseases are also provided. Published 2019. Compr Physiol 9:1443-1502, 2019., (Copyright © 2019 American Physiological Society. All rights reserved.)

Exposing adrenal chromaffin cells to single 150 to 400 ns electric pulses triggers a rise in intracellular Ca 2+ ([Ca 2+ ] i ) that is due to Ca 2+ influx through voltage-gated Ca 2+ channels (VGCC) and plasma membrane electropores. Immediate delivery of a second pulse of the opposite polarity in which the duration and amplitude were the same as the first pulse (a symmetrical bipolar pulse) or greater than the first pulse (an asymmetrical bipolar pulse) had a stimulatory effect, evoking larger Ca 2+ responses than the corresponding unipolar pulse. Progressively decreasing the amplitude of the opposite polarity pulse while also increasing its duration converted stimulation to attenuation, which reached a maximum of 43% when the positive phase was 150 ns at 3.1 kV/cm, and the negative phase was 800 ns at 0.2 kV/cm. When VGCCs were blocked, Ca 2+ responses evoked by asymmetrical and even symmetrical bipolar pulses were significantly reduced relative to those evoked by the corresponding unipolar pulse under the same conditions, indicating that attenuation involved mainly the portion of Ca 2+ influx attributable to membrane electropermeabilization. Thus, by tuning the shape of the bipolar pulse, Ca 2+ entry into chromaffin cells through electropores could be attenuated while preserving Ca 2+ influx through VGCCs.

Adenosine triphosphate (ATP) is the main energy source for cellular metabolism. Besides that, ATP is a neurotransmitter and a cotransmitter that acts on purinergic receptors present either pre- or postsynaptically. Almost all types of secretory vesicles from any neuron or animal species contain high concentrations of ATP, being an essential factor in the accumulation of neurotransmitters. In this work, we studied the effects of ATP on quantum catecholamine release and vesicular storage in chromaffin cells. We combined three electrochemical methods: conventional amperometry with intracellular vesicle impact electrochemical cytometry and vesicle impact electrochemical cytometry. We found that extracellular ATP increased the released quantal fraction of catecholamine but not its vesicular content. Studying the dynamics of exocytosis events in ATP treated cells showed that ATP affects the release fusion pore. To elucidate the mechanisms of the observed ATP effects, cells and vesicles were pharmacologically treated with suramin (a purinergic blocker) and ARL-67156 (an antagonist of ecto-ATPases). The data indicate that the catecholamine content of vesicles increased compared to control after these drugs. Our data suggest that ATP acting on purinergic receptors increases the quantum releasable size through an increased fusion pore opening and that ARL-67156 and/or suramin protect the vesicle from neurotransmitter leakage by functioning as competitive inhibitors to ATP.

Key Points: Tymothy syndrome (TS) is a multisystem disorder featuring cardiac arrhythmias, autism and adrenal gland dysfunction that originates from a de novo point mutation in the gene encoding the Cav1.2 (CACNA1C) L-type channel. To study the role of Cav1.2 channel signals in autism, the autistic TS2-neo mouse has been generated bearing the G406R point-mutation associated with TS type-2. Using heterozygous TS2-neo mice, we report that the G406R mutation reduces the rate of inactivation and shifts leftward the activation and inactivation of L-type channels, causing marked increase of resting Ca 2+ influx ('window' Ca 2+ current). The increased 'window current' causes marked reduction of Na V channel density, switches normal tonic firing to abnormal burst firing, reduces mitochondrial metabolism, induces cell swelling and decreases catecholamine release. Overnight incubations with nifedipine rescue Na V channel density, normal firing and the quantity of catecholamine released. We provide evidence that chromaffin cell malfunction derives from altered Cav1.2 channel gating., Abstract: L-type voltage-gated calcium (Cav1) channels have a key role in long-term synaptic plasticity, sensory transduction, muscle contraction and hormone release. A point mutation in the gene encoding Cav1.2 (CACNA1C) causes Tymothy syndrome (TS), a multisystem disorder featuring cardiac arrhythmias, autism spectrum disorder (ASD) and adrenal gland dysfunction. In the more severe type-2 form (TS2), the missense mutation G406R is on exon 8 coding for the IS6-helix of the Cav1.2 channel. The mutation causes reduced inactivation and induces autism. How this occurs and how Cav1.2 gating-changes alter cell excitability, neuronal firing and hormone release on a molecular basis is still largely unknown. Here, using the TS2-neo mouse model of TS we show that the G406R mutation altered excitability and reduced secretory activity in adrenal chromaffin cells (CCs). Specifically, the TS2 mutation reduced the rate of voltage-dependent inactivation and shifted leftward the activation and steady-state inactivation of L-type channels. This markedly increased the resting 'window' Ca 2+ current that caused an increased percentage of CCs undergoing abnormal action potential (AP) burst firing, cell swelling, reduced mitochondrial metabolism and decreased catecholamine release. The increased 'window' Ca 2+ current caused also decreased Na V channel density and increased steady-state inactivation, which contributed to the increased abnormal burst firing. Overnight incubation with the L-type channel blocker nifedipine rescued the normal AP firing of CCs, the density of functioning Na V channels and their steady-state inactivation. We provide evidence that CC malfunction derives from the altered Cav1.2 channel gating and that dihydropyridines are potential therapeutics for ASD., (© 2019 University of Torino. The Journal of Physiology © 2019 The Physiological Society.)

Background: Quantal exocytosis of oxidizable neurotransmitters can be detected as spikes of amperometric current using electrochemical microelectrodes. Measurements of spike parameters indicate the maximal transmitter flux, flux duration, and amount of transmitter released from individual vesicles. Automated analysis algorithms need to reject spikes that overlap in time. In addition, many spikes are preceded by small amplitude "foot" signals, attributed to slow release of transmitter through a fusion pore. Accurate pre-spike baseline determination is essential for estimating fusion-pore duration and the amount of transmitter released through the fusion pore., New Method: We developed an estimation approach that is based on fitting a multi-exponential function to the data. Our previously described matched-filter algorithm is used to identify the sections of data to fit and provides seed values to facilitate convergence of the iterative fit. The new estimation algorithm includes overlap rejection, a two-step fitting procedure and a novel baseline estimation procedure., Results: Histograms of spike parameters demonstrate excellent agreement of the new approach with manually computed parameters., Comparison With Existing Methods: Parameter estimates generated using the new approach are closer to blind manual estimates than commonly used existing methods. The improved performance is due to better detection of valid spikes and rejection of overlapping spikes. Moreover, since the complete time course of the spike is fit to a function, more complete information about the spike time course is captured., Conclusions: The matched-filter seeded algorithm reliably rejects overlaps and estimates spike and foot signal parameters in a fully automated manner., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Chromaffin cells from the adrenal gland (CCs) have extensively been used to explore the molecular structure and function of the exocytotic machinery, neurotransmitter release and synaptic transmission. The CC is integrated in the sympathoadrenal axis that helps the body maintain homoeostasis during both routine life and in acute stress conditions. This function is exquisitely controlled by the cerebral cortex and the hypothalamus. We propose the hypothesis that damage undergone by the brain during neurodegenerative diseases is also affecting the neurosecretory function of adrenal medullary CCs. In this context, we review here the following themes: (i) How the discharge of catecholamines is centrally and peripherally regulated at the sympathoadrenal axis; (ii) which are the intricacies of the amperometric techniques used to study the quantal release of single-vesicle exocytotic events; (iii) which are the alterations of the exocytotic fusion pore so far reported, in CCs of mouse models of neurodegenerative diseases; (iv) how some proteins linked to neurodegenerative pathologies affect the kinetics of exocytotic events; (v) finally, we try to integrate available data into a hypothesis to explain how the centrally originated neurodegenerative diseases may alter the kinetics of single-vesicle exocytotic events in peripheral adrenal medullary CCs., (© 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Three divalent cations can elicit secretory responses in most neuroendocrine cells, including chromaffin cells. The extent to which secretion is elicited by the cations in intact depolarized cells was Ba 2+  > Sr 2+  ≥ Ca 2+ , contrasting with that elicited by these cations in permeabilized cells (Ca 2+  > Sr 2+  > Ba 2+ ). Current-clamp recordings show that extracellular Sr 2+ and Ba 2+ cause membrane depolarization and action potentials, which are not blocked by Cd 2+ but that can be mimicked by tetra-ethyl-ammonium. When applied intracellularly, only Ba 2+ provokes action potentials. Voltage-clamp monitoring of Ca 2+ -activated K + channels (K Ca ) shows that Ba 2+ reduces outward currents, which were enhanced by Sr 2+ . Extracellular Ba 2+ increases cytosolic Ca 2+ concentrations in Fura-2-loaded intact cells, and it induces long-lasting catecholamine release. Conversely, amperometric recordings of permeabilized cells show that Ca 2+ promotes the longest lasting secretion, as Ba 2+ only provokes secretion while it is present and Sr 2+ induces intermediate-lasting secretion. Intracellular Ba 2+ dialysis provokes exocytosis at concentrations 100-fold higher than those of Ca 2+ , whereas Sr 2+ exhibits an intermediate sensitivity. These results are compatible with the following sequence of events: Ba 2+ blocks K Ca channels from both the outside and inside of the cell, causing membrane depolarization that, in turn, opens voltage-sensitive Ca 2+ channels and favors the entry of Ca 2+ and Ba 2+ . Although Ca 2+ is less permeable through its own channels, it is more efficient in triggering exocytosis. Strontium possesses both an intermediate permeability and an intermediate ability to induce secretion.

Chromaffin cells from the adrenal medulla participate in stress responses by releasing catecholamines into the bloodstream. Main control of adrenal catecholamine secretion is exerted both neurally (by the splanchnic nerve fibers) and humorally (by corticosteroids, circulating noradrenaline, etc.). It should be noted, however, that secretory products themselves (catecholamines, ATP, opioids, ascorbic acid, chromogranins) could also influence the secretory response in an autocrine/paracrine manner. This form of control is activity-dependent and can be either inhibitory or excitatory. Among the inhibitory influences, it stands out the one mediated by α 2 -adrenergic autoreceptors activated by released catecholamines. α 2 -adrenoceptors are G protein-coupled receptors capable to inhibit exocytotic secretion through a direct interaction of Gβγ subunits with voltage-gated Ca 2+ channels. Interestingly, upon intense and/or prolonged stimulation, α 2 -adrenergic receptors become desensitized by the intervention of G protein-coupled receptor kinase 2 (GRK2). In several experimental models of heart failure, there has been reported the up-regulation of GRK2 and the loss of functioning of inhibitory α 2 -adrenoceptors resulting in enhanced release of adrenomedullary catecholamines. Given the importance of circulating catecholamines in the pathophysiology of heart failure, the recovery of α 2 -adrenergic modulation of the secretory response from chromaffin cells appears as a novel strategy for a better control of the patients with this cardiac disease.

Amperometry is a powerful method to record quantal release events from chromaffin cells and is widely used to assess how specific drugs modify quantal size, kinetics of release, and early fusion pore properties. Surface-modified CMOS-based electrochemical sensor arrays allow simultaneous recordings from multiple cells. A reliable, low-cost technique is presented here for efficient targeting of single cells specifically to the electrode sites. An SU-8 microwell structure is patterned on the chip surface to provide insulation for the circuitry as well as cell trapping at the electrode sites. A shifted electrode design is also incorporated to increase the flexibility of the dimension and shape of the microwells. The sensitivity of the electrodes is validated by a dopamine injection experiment. Microwells with dimensions slightly larger than the cells to be trapped ensure excellent single-cell targeting efficiency, increasing the reliability and efficiency for on-chip single-cell amperometry measurements. The surface-modified device was validated with parallel recordings of live chromaffin cells trapped in the microwells. Rapid amperometric spikes with no diffusional broadening were observed, indicating that the trapped and recorded cells were in very close contact with the electrodes. The live cell recording confirms in a single experiment that spike parameters vary significantly from cell to cell but the large number of cells recorded simultaneously provides the statistical significance.

The role of gamma-aminobutyric acid (GABA) in adrenal medulla chromaffin cell (CC) function is just beginning to unfold. GABA is stored in catecholamine (CA)-containing dense core granules and is presumably released together with CA, ATP, and opioids in response to physiological stimuli, playing an autocrine-paracrine role on CCs. The reported paradoxical "dual action" of GABA A -R activation (enhancement of CA secretion and inhibition of synaptically evoked CA release) is only one aspect of GABA's multifaceted actions. In this review, we discuss recent physiological experiments on rat CCs in situ which suggest that GABA regulation of CC function may depend on the physiological context: During non-stressful conditions, GABA A -R activation by endogenous GABA tonically inhibits acetylcholine release from splanchnic nerve terminals and decreases spontaneous Ca 2+ fluctuations in CCs, preventing unwanted CA secretion. During intense stress, splanchnic nerve terminals release acetylcholine, which depolarizes CCs and allows the Ca 2+ influx that triggers the release of CA and GABA. With time, CA secretion declines, due to voltage-independent inhibition of Ca 2+ channels and desensitization of cholinergic nicotinic receptors. Nonetheless, acute activation of GABA A -R is depolarizing in about 50% of CCs, and thus GABA, acting as an autocrine/paracrine mediator, could help to maintain CA exocytosis under stress. GABA A -R activation is not excitatory in about half of CCs' population because it hyperpolarizes them or elicits no response. This percentage possibly varies, depending on functional demands, since GABA A -R-mediated actions are determined by the intracellular chloride concentration ([Cl - ] i ) and therefore on the activity of cation-chloride co transporters, which is functionally regulated. These findings underscore a potential importance of a novel and complex GABA-mediated regulation of CC function and of CA secretion.

Background: Electrochemical microelectrodes located immediately adjacent to the cell surface can detect spikes of amperometric current during exocytosis as the transmitter released from a single vesicle is oxidized on the electrode surface. Automated techniques to detect spikes are needed in order to quantify the spike rate as a measure of the rate of exocytosis., New Method: We have developed a Matched Filter (MF) detection algorithm that scans the data set with a library of prototype spike templates while performing a least-squares fit to determine the amplitude and standard error. The ratio of the fit amplitude to the standard error constitutes a criterion score that is assigned for each time point and for each template. A spike is detected when the criterion score exceeds a threshold and the highest-scoring template and the time of peak score is identified. The search for the next spike commences only after the score falls below a second, lower threshold to reduce false positives. The approach was extended to detect spikes with double-exponential decays with the sum of two templates., Results: Receiver Operating Characteristic plots (ROCs) demonstrate that the algorithm detects >95% of manually identified spikes with a false-positive rate of ∼2%., Comparison With Existing Methods: ROCs demonstrate that the MF algorithm performs better than algorithms that detect spikes based on a derivative-threshold approach., Conclusions: The MF approach performs well and leads into approaches to identify spike parameters., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Pituitary adenylate cyclase-activating polypeptide (PACAP) was first identified in hypothalamus, based on its ability to elevate cyclic AMP in the anterior pituitary. PACAP has been identified as the adrenomedullary neurotransmitter in stress through a combination of ex vivo, in vivo, and in cellula experiments over the past two decades. PACAP causes catecholamine secretion, and activation of catecholamine biosynthetic enzymes, during episodes of stress in mammals. Features of PACAP signaling allowing stress transduction at the splanchnicoadrenomedullary synapse have yielded insights into the contrasting roles of acetylcholine's and PACAP's actions as first messengers at the chromaffin cell, via differential release at low and high rates of splanchnic nerve firing, and differential signaling pathway engagement leading to catecholamine secretion and chromaffin cell gene transcription. Secretion stimulated by PACAP, via calcium influx independent of action potential generation, is under active investigation in several laboratories both at the chromaffin cell and within autonomic ganglia of both the parasympathetic and sympathetic nervous systems. PACAP is a neurotransmitter important in stress transduction in the central nervous system as well, and is found at stress-transduction nuclei in brain including the paraventricular nucleus of hypothalamus, the amygdala and extended amygdalar nuclei, and the prefrontal cortex. The current status of PACAP as a master regulator of stress signaling in the nervous system derives fundamentally from the establishment of its role as the splanchnicoadrenomedullary transmitter in stress. Experimental elucidation of PACAP action at this synapse remains at the forefront of understanding PACAP's role in stress signaling throughout the nervous system.

During the last 10 years, we have been working on human chromaffin cells obtained from the adrenal gland of organ donors that suffered encephalic or cardiac death. We first electrophysiologically characterized the nicotinic acetylcholine receptors (nAChRs) activated by acetylcholine, and their contribution to the exocytosis of chromaffin vesicles and release of catecholamines. We have shown that these cells possess an adrenergic phenotype. This phenotype may contribute to an increased expression of α7 nAChRs in these cells, allowing for recording of α7 nAChR currents, something that had previously not been achieved in non-human species. The use of α-conotoxins allowed us to characterize non-α7 nAChR subtypes and, together with molecular biology experiments, conclude that the predominant nAChR subtype in human chromaffin cells is α3β4* (asterisk indicates the posible presence of additional subunits). In addition, there is a minor population of αxβ2 nAChRs. Both α7 and non-α7 nAChR subtypes contribute to the exocytotic process. Exocytosis mediated by nAChRs could be as large in magnitude as that elicited by calcium entry through voltage-dependent calcium channels. Finally, we have also investigated the effect of nAChR-targeted tobacco cessation drugs on catecholamine release in chromaffin cells. We have concluded that at therapeutic concentrations, varenicline alone does not increase the frequency of action potentials evoked by ACh. However, varenicline in the presence of nicotine does increase this frequency, and thus, in the presence of both drugs, the probability of increased catecholamine release in human chromaffin cells is high.

From birth to death, catecholamine secretion undergoes continuous adjustments, allowing the organism to adapt to homeostasis changes. To cope with these stressful conditions, the neuroendocrine cells of the adrenal medulla play an immediate and crucial role. Chromaffin cell-driven catecholamine release is chiefly controlled by a neurogenic command that arises from the sympathetic nervous system, which releases acetylcholine at the splanchnic nerve terminal-chromaffin cell synapses. In addition to receiving several synaptic inputs individually, chromaffin cells are coupled by gap junctions. This raises interesting questions about the usefulness and the role of the gap junctional coupling within the chromaffin tissue, considering that secretory function is efficiently completed by the neurogenic pathway. The findings that gap junctions contribute to catecholamine secretion, both ex vivo and in vivo, provide some early answers, but their involvement in other cellular functions still remains unexplored. This review summarizes the molecular and physiological evidence that gap junctions can act either as an accelerator or a brake of stimulus-secretion coupling and discusses this functional plasticity in the context of specific needs in circulating catecholamine levels. It introduces the concept of gap junctions as sympathetic activity sensors and guardians of the functional integrity of the chromaffin tissue.

The coexistence of different subtypes of voltage-dependent calcium channels (VDCC) within the same chromaffin cell (CC) and the marked interspecies variability in the proportion of VDCC subtypes that are present in the plasmalemma of the CCs raises the question on their roles in controlling different physiological functions. Particularly relevant seems to be the role of VDCCs in the regulation of the exocytotic neurotransmitter release process, and its tightly coupled membrane retrieval (endocytosis) process since both are Ca 2+ -dependent processes. This review is focused on the role of Ca 2+ influx through L-type VDCC in the regulation of these two processes. It is currently accepted that the different VDCC subtypes (i.e., T, L, N, P/Q, R) contribute to exocytosis proportionally to their density of expression and gating properties. However, the pattern of stimulation defines a preferential role of the different subtypes of VDCC on exocytosis and endocytosis. Thus, L-type channels seem to control catecholamine release induced by prolonged stimuli while fast exocytosis in response to short square depolarizing pulses or action potentials is mediated by Ca 2+ entering CCs through P/Q channels. The pattern of stimulation also influences the endocytotic process, and thus, electrophysiological data suggest the sustained Ca 2+ entry through slow-inactivating L-type channels could be responsible for the activation of fast endocytosis.

Adrenal medullary chromaffin cells in mammals are innervated by sympathetic preganglionic nerve fibers, as are sympathetic ganglion neurons. Acetylcholine in the ganglion neurons is well established as mediating fast and slow excitatory postsynaptic potentials through nicotinic and muscarinic acetylcholine receptors (AChRs), respectively. The role of muscarinic AChRs during neuronal transmission in chromaffin cells varies among different mammals. Furthermore, the ion channel mechanisms associated with the muscarinic AChR-mediated increase in excitability of chromaffin cells are complicated and different from the excitation of ganglion neurons, which has been ascribed to the inhibition of M-type K + channels. In this review, we focus on muscarinic receptor-mediated excitation in rodent and guinea pig chromaffin cells, in particular, on the role of muscarinic receptors in neuronal transmission, the muscarinic receptor subtypes involved in excitation and secretion, and the muscarinic regulation of ion channels including TWIK-related acid-sensitive K + channels. Finally, we discuss prospectively the future of muscarinic receptor research in adrenal chromaffin cells.

Adrenal chromaffin cells (CCs) are the main source of circulating catecholamines (CAs) that regulate the body response to stress. Release of CAs is controlled neurogenically by the activity of preganglionic sympathetic neurons through trains of action potentials (APs). APs in CCs are generated by robust depolarization following the activation of nicotinic and muscarinic receptors that are highly expressed in CCs. Bovine, rat, mouse, and human CCs also express a composite array of Na + , K + , and Ca 2+ channels that regulate the resting potential, shape the APs, and set the frequency of AP trains. AP trains of increasing frequency induce enhanced release of CAs. If the primary role of CCs is simply to relay preganglionic nerve commands to CA secretion, why should they express such a diverse set of ion channels? An answer to this comes from recent observations that, like in neurons, CCs undergo complex firing patterns of APs suggesting the existence of an intrinsic CC excitability (non-neurogenically controlled). Recent work has shown that CCs undergo occasional or persistent burst firing elicited by altered physiological conditions or deletion of pore-regulating auxiliary subunits. In this review, we aim to give a rationale to the role of the many ion channel types regulating CC excitability. We will first describe their functional properties and then analyze how they contribute to pacemaking, AP shape, and burst waveforms. We will also furnish clear indications on missing ion conductances that may be involved in pacemaking and highlight the contribution of the crucial channels involved in burst firing.

The protein α-synuclein has a central role in the pathogenesis of Parkinson's disease. Like that of other proteins that accumulate in neurodegenerative disease, however, the function of α-synuclein remains unknown. Localization to the nerve terminal suggests a role in neurotransmitter release, and overexpression inhibits regulated exocytosis, but previous work has failed to identify a clear physiological defect in mice lacking all three synuclein isoforms. Using adrenal chromaffin cells and neurons, we now find that both overexpressed and endogenous synuclein accelerate the kinetics of individual exocytotic events, promoting cargo discharge and reducing pore closure ('kiss-and-run'). Thus, synuclein exerts dose-dependent effects on dilation of the exocytotic fusion pore. Remarkably, mutations that cause Parkinson's disease abrogate this property of α-synuclein without impairing its ability to inhibit exocytosis when overexpressed, indicating a selective defect in normal function.

Key Points: Upon repeated application of short ACh pulses to C57BL6J mouse chromaffin cells, the amperometrically monitored secretory responses promptly decayed to a steady-state level of around 25% of the initial response. A subsequent K + pulse, however, overcame such decay. These data suggest that mouse chromaffin cells have a ready release-vesicle pool that is selectively recruited by the physiological neurotransmitter ACh. The ACh-sensitive vesicle pool is refilled and maintained by the rate of Ca 2+ delivery from mitochondria to the cytosol, through the mitochondrial Na + /Ca 2+ exchanger (mNCX). ITH12662, a novel blocker of the mNCX, prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca 2+ ] c clearance. This regulatory pathway may be physiologically relevant in situations of prolonged stressful conflicts where a sustained catecholamine release is regulated by mitochondrial Ca 2+ circulation through the mNCX, which couples respiration and ATP synthesis to long-term stimulation of chromaffin cells by endogenously released ACh., Abstract: Using caged-Ca 2+ photorelease or paired depolarising pulses in voltage-clamped chromaffin cells (CCs), various pools of secretory vesicles with different readiness to undergo exocytosis have been identified. Whether these pools are present in unclamped CCs challenged with ACh, the physiological neurotransmitter at the splanchnic nerve-CC synapse, is unknown. We have explored here whether an ACh-sensitive ready-release vesicle pool (ASP) is present in C57BL6J mouse chromaffin cells (MCCs). Single cells were fast perfused with a Tyrode solution at 37°C, and challenged with 12 sequential ACh pulses (100 μm, 2 s, every 30 s) plus a K + pulse given at the end (75 mm K + ). After the first 2-3 ACh pulses the amperometrically monitored secretory responses promptly decayed to a steady-state level of around 25% of the initial response. The last K + pulse, however, overcame such decay. Repeated ACh pulses to voltage-clamped cells elicited non-desensitising nicotinic currents. Also, the [Ca 2+ ] c transients elicited by repeated ACh pulses that were superimposed on a stable baseline elevation did not undergo decay. The novel blocker of the mitochondrial Na + /Ca 2+ exchanger (mNCX) ITH12662 prevented the decay of secretion elicited by ACh pulses and delayed the rate of [Ca 2+ ] c clearance. The experiments are compatible with the idea that C57BL6J MCCs have an ASP vesicle pool that is selectively recruited by the physiological neurotransmitter ACh and is regulated by the rate of Ca 2+ delivery from mitochondria to the cytosol, through the mNCX., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Varenicline is a nicotinic acetylcholine receptor (nAChR) agonist used to treat nicotine addiction, but a live debate persists concerning its mechanism of action in reducing nicotine consumption. Although initially reported as α4β2 selective, varenicline was subsequently shown to activate other nAChR subtypes implicated in nicotine addiction including α3β4. However, it remains unclear whether activation of α3β4 nAChRs by therapeutically relevant concentrations of varenicline is sufficient to affect the behavior of cells that express this subtype. We used patch-clamp electrophysiology to assess the effects of varenicline on native α3β4* nAChRs (asterisk denotes the possible presence of other subunits) expressed in human adrenal chromaffin cells and compared its effects to those of nicotine. Varenicline and nicotine activated α3β4* nAChRs with EC 50 values of 1.8 (1.2-2.7) μM and 19.4 (11.1-33.9) μM, respectively. Stimulation of adrenal chromaffin cells with 10 ms pulses of 300 μM acetylcholine (ACh) in current-clamp mode evoked sodium channel-dependent action potentials (APs). Under these conditions, perfusion of 50 or 100 nM varenicline showed very little effect on AP firing compared to control conditions (ACh stimulation alone), but at higher concentrations (250 nM) varenicline increased the number of APs fired up to 436 ± 150%. These results demonstrate that therapeutic concentrations of varenicline are unlikely to alter AP firing in chromaffin cells. In contrast, nicotine showed no effect on AP firing at any of the concentrations tested (50, 100, 250, and 500 nM). However, perfusion of 50 nM nicotine simultaneously with 100 nM varenicline increased AP firing by 290 ± 104% indicating that exposure to varenicline and nicotine concurrently may alter cellular behavior such as excitability and neurotransmitter release., (© 2016 International Society for Neurochemistry.)

Patch clamp electrophysiology serves as a powerful method for studying changes in plasma membrane ion conductance induced by externally applied high-intensity nanosecond electric pulses (NEPs). This paper describes an enhanced monitoring technique that minimizes the length of time between pulse exposure and data recording in a patch-clamped excitable cell. Whole-cell membrane currents were continuously recorded up to 11 ms before and resumed 8 ms after delivery of a 5-ns, 6 MV/m pulse by a pair of tungsten rod electrodes to a patched adrenal chromaffin cell maintained at a holding potential of -70 mV. This timing was achieved by two sets of relay switches. One set was used to disconnect the patch pipette electrode from the pre-amplifier and connect it to a battery to maintain membrane potential at -70 mV, and also to disconnect the reference electrode from the amplifier. The other set was used to disconnect the electrodes from the pulse generator until the time of NEP/sham exposure. The sequence and timing of both sets of relays were computer-controlled. Using this procedure, we observed that a 5-ns pulse induced an instantaneous inward current that decayed exponentially over the course of several minutes, that a second pulse induced a similar response, and that the current was carried, at least in part, by Na + . This approach for characterizing ion conductance changes in an excitable cell in response to NEPs will yield information essential for assessing the potential use of NEP stimulation for therapeutic applications.

Annexin A2 is a calcium-, actin-, and lipid-binding protein implicated in exocytosis in different cell types, such as neuroendocrine cells. In chromaffin cells, cytosolic annexin A2 is recruited to the plasma membrane upon cell stimulation. Here, we review the latest evidence detailing the functional importance of annexin A2 in different stages of exocytosis. These include the recruitment of annexin A2 to the plasma membrane near soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes, the role of annexin A2 in the formation of lipid domains at exocytotic sites, and finally the annexin A2 bundling of actin microfilaments associated with chromaffin granules. These structures induce first the coalescence of lipid domains required for the formation of the exocytotic site, and in the second time, exert mechanical force on the granule to favor fusion pore expansion and squeeze the granule to facilitate catecholamine release. Annexin A2 is a calcium-, actin-, and lipid-binding protein implicated in exocytosis in different cell types, including neuroendocrine cells. Upon cell stimulation, annexin A2 translocates from the cytosol to the plasma membrane of chromaffin cells and bundles actin filaments associated with chromaffin granules. This promotes the formation of lipid domains required for granule docking, and facilitates catecholamine release by compressing the granule. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015)., (© 2016 International Society for Neurochemistry.)

Using fluorescence [Ca(2+)]i imaging in rat adrenal slices, we characterized the effects of agonists and antagonists of the GABAA receptor (GABAA-R) on resting intracellular Ca(2+) ([Ca(2+)]i) and spontaneous [Ca(2+)]i fluctuations (SCFs) in hundreds of individual chromaffin cells (CCs) recorded simultaneously in situ. Muscimol, a GABAA-R agonist (20 μM; 25 s), induced an increase of resting [Ca(2+)]i in 43 ± 3 % of CCs, a decrease in 26 ± 2 %, and no response in 30 ± 5 %. In Ca(2+)-free external medium, SCFs ceased completely and muscimol failed to elicit [Ca(2+)]i rises. All muscimol-induced [Ca(2+)]i changes were blocked by the GABAA-R antagonist bicuculline, suggesting that they result from changes in membrane potential depending on the cell's Cl(-) equilibrium potential. Unexpectedly, bicuculline increased the amplitude and frequency of SCFs in 54 % of CCs, revealing a tonic inhibition of SCFs by ambient GABA acting through GABAA-R. Mecamylamine (a specific nicotinic cholinergic blocker) decreased basal SCF activity in 18 % of CCs and inhibited bicuculline-induced SCF intensification, suggesting that spontaneous acetylcholine (ACh) release from nerve endings contributes to SCF generation in CCs in situ and that blockade of presynaptic GABAA-Rs intensifies SCFs in part through the disinhibition of spontaneous cholinergic transmission. Electrophysiological experiments confirmed that spontaneous excitatory postsynaptic currents recorded from CCs in situ were enhanced by bicuculline. To our knowledge, this is the first description of a regulatory effect of endogenous GABA on synaptic currents and SCFs of adrenal CCs. These findings denote a novel GABA-mediated presynaptic and postsynaptic regulatory mechanism of CC activity which may participate in the control of catecholamine secretion.

Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles' arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that including the fusion-time statistics in our model does not produce any significant changes on the results. These findings indicate that the motion of the whole ensemble of vesicles towards the membrane is directed and reflected in the amperometric signals. Our results confirm the conclusions of previous imaging studies performed on single vesicles that vesicles' motion underneath plasma membranes is not purely random, but biased towards the membrane.

In search of druggable synthetic lipids that function as potential modulators of synaptic transmission and plasticity, we synthesized sulfoglycolipid IG20, which stimulates neuritic outgrowth. Here, we have explored its effects on ion channels and exocytosis in bovine chromaffin cells. IG20 augmented the rate of basal catecholamine release. Such effect did not depend on Ca(2+) mobilization from intracellular stores; rather, IG20-elicited secretion entirely dependent on Ca(2+) entry through L-subtype voltage-activated Ca(2+) channels. Those channels were recruited by cell depolarization mediated by IG20 likely through its ability to enhance the recruitment of Na(+) channels at more hyperpolarizing potentials. Confocal imaging with fluorescent derivative IG20-NBD revealed its rapid incorporation and confinement into the plasmalemma, supporting the idea that IG20 effects are exerted through a plasmalemmal-delimited mechanism. Thus, synthetic IG20 seems to mimic several physiological effects of endogenous lipids such as regulation of ion channels, Ca(2+) signaling, and exocytosis. Therefore, sulfoglycolipid IG20 may become a pharmacological tool for investigating the role of the lipid environment on neuronal excitability, ion channels, neurotransmitter release, synaptic efficacy, and neuronal plasticity. It may also inspire the synthesis of druggable sulfoglycolipids aimed at increasing synaptic plasticity and efficacy in neurodegenerative diseases and traumatic brain-spinal cord injury. The novel synthetic sulfoglycolipid IG20 mimics several physiological effects of endogenous lipids such as regulation of ion channels, Ca(2+) signaling, and exocytosis. This profile may eventually drive enhanced synaptic plasticity and efficacy., (© 2015 International Society for Neurochemistry.)

Key Points: Leptin is an adipokine produced by the adipose tissue regulating body weight through its appetite-suppressing effect and, as such, exerts a relevant action on the adipo-adrenal axis. Leptin has a dual action on adrenal mouse chromaffin cells both at rest and during stimulation. At rest, the adipokine inhibits the spontaneous firing of most cells by enhancing the probability of BK channel opening through the phosphoinositide 3-kinase signalling cascade. This inhibitory effect is absent in db(-) /db(-) mice deprived of Ob receptors. During sustained stimulation, leptin preserves cell excitability by generating well-adapted action potential (AP) trains of lower frequency and broader width and increases catecholamine secretion by increasing the size of the ready-releasable pool and the rate of vesicle release. In conclusion, leptin dampens AP firing at rest but preserves AP firing and enhances catecholamine release during sustained stimulation, highlighting the importance of the adipo-adrenal axis in the leptin-mediated increase of sympathetic tone and catecholamine release., Abstract: Leptin is an adipokine produced by the adipose tissue regulating body weight through its appetite-suppressing effect. Besides being expressed in the hypothalamus and hippocampus, leptin receptors (ObRs) are also present in chromaffin cells of the adrenal medulla. In the present study, we report the effect of leptin on mouse chromaffin cell (MCC) functionality, focusing on cell excitability and catecholamine secretion. Acute application of leptin (1 nm) on spontaneously firing MCCs caused a slowly developing membrane hyperpolarization followed by complete blockade of action potential (AP) firing. This inhibitory effect at rest was abolished by the BK channel blocker paxilline (1 μm), suggesting the involvement of BK potassium channels. Single-channel recordings in 'perforated microvesicles' confirmed that leptin increased BK channel open probability without altering its unitary conductance. BK channel up-regulation was associated with the phosphoinositide 3-kinase (PI3K) signalling cascade because the PI3K specific inhibitor wortmannin (100 nm) fully prevented BK current increase. We also tested the effect of leptin on evoked AP firing and Ca(2+) -driven exocytosis. Although leptin preserves well-adapted AP trains of lower frequency, APs are broader and depolarization-evoked exocytosis is increased as a result of the larger size of the ready-releasable pool and higher frequency of vesicle release. The kinetics and quantal size of single secretory events remained unaltered. Leptin had no effect on firing and secretion in db(-) /db(-) mice lacking the ObR gene, confirming its specificity. In conclusion, leptin exhibits a dual action on MCC activity. It dampens AP firing at rest but preserves AP firing and increases catecholamine secretion during sustained stimulation, highlighting the importance of the adipo-adrenal axis in the leptin-mediated increase of sympathetic tone and catecholamine release., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Mitochondria regulate cellular Ca(2+) oscillations, taking up Ca(2+) through its uniporter and releasing it through the mitochondrial sodium/calcium exchanger. The role of mitochondria in the regulation of Ca(2+) cycle has received much attention recently, as it is a central stage in neuronal survival and death processes. Over the last decades, the 4,1-benzothiazepine CGP37157 has been the only available blocker of the mitochondrial sodium/calcium exchanger, although it targets several other calcium transporters. We report the synthesis of 4,1-benzothiazepine derivatives with the goal of enhancing mitochondrial sodium/calcium exchanger blockade and selectivity, and the evaluation of their cytoprotective effect. The compound 4c presented an interesting neuroprotective profile in addition to an important blockade of the mitochondrial sodium/calcium exchanger. The use of this benzothiazepine could help to understand the physiological functions of the mitochondrial sodium/calcium exchanger. In addition, we hypothesize that a moderate blockade of the mitochondrial sodium/calcium exchanger would provide enhanced neuroprotection in neurons.

Annexin A2, a calcium-, actin-, and lipid-binding protein involved in exocytosis, mediates the formation of lipid microdomains required for the structural and spatial organization of fusion sites at the plasma membrane. To understand how annexin A2 promotes this membrane remodeling, the involvement of cortical actin filaments in lipid domain organization was investigated. 3D electron tomography showed that cortical actin bundled by annexin A2 connected docked secretory granules to the plasma membrane and contributed to the formation of GM1-enriched lipid microdomains at the exocytotic sites in chromaffin cells. When an annexin A2 mutant with impaired actin filament-bundling activity was expressed, the formation of plasma membrane lipid microdomains and the number of exocytotic events were decreased and the fusion kinetics were slower, whereas the pharmacological activation of the intrinsic actin-bundling activity of endogenous annexin A2 had the opposite effects. Thus, annexin A2-induced actin bundling is apparently essential for generating active exocytotic sites., (© 2015 Gabel et al.)

The effects of mitochondrial inhibitors (CN(-), a complex IV inhibitor and CCCP, protonophore) on catecholamine (CA) secretion and mitochondrial function were explored functionally and biochemically in rat and guinea-pig adrenal chromaffin cells. Guinea-pig chromaffin cells conspicuously secreted CA in response to CN(-) or CCCP, but rat cells showed a little, if any, secretory response to either of them. The resting metabolic rates in rat adrenal medullae did not differ from those in guinea-pig adrenal medullae. On the other hand, the time course of depolarization of the mitochondrial membrane potential (ΔΨm) in guinea-pig chromaffin cells in response to CN(-) was slower than that in rat chromaffin cells, and this difference was abolished by oligomycin, an F1F0-ATPase inhibitor. The extent of CCCP-induced decrease in cellular ATP in guinea-pig chromaffin cells, which was indirectly measured using a Mg(2+) indicator, was smaller than that in rat chromaffin cells. Relative expression levels of F1F0-ATPase inhibitor factor in guinea-pig adrenal medullae were smaller than in rat adrenal medullae, and the opposite was true for F1F0-ATPase α subunit. The present results indicate that guinea-pig chromaffin cells secrete more CA in response to a mitochondrial inhibitor than rat chromaffin cells and this higher susceptibility in the former is accounted for by a larger extent of reversed operation of F1F0-ATPase with the consequent decrease in ATP under conditions where ΔΨm is depolarized., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Membrane fusion requires that nearly flat lipid bilayers deform into shapes with very high curvature. This makes membrane bending a critical force in determining fusion mechanisms. A lipid bilayer will bend spontaneously when material is distributed asymmetrically between its two monolayers, and its spontaneous curvature (C0) will influence the stability of curved fusion intermediates. Prior work on Ca(2+)-triggered exocytosis revealed that fusion pore lifetime (τ) varies with vesicle content (Q), and showed that this relation reflects membrane bending energetics. Lipids that alter C0 change the dependence of τ on Q. These results suggested that the greater stability of an initial exocytotic fusion pore associated with larger vesicles reflects the need to bend more membrane during fusion pore dilation. In this study, we explored the possibility of manipulating C0 by mutating the transmembrane domain (TMD) of the vesicle membrane protein synaptobrevin 2 (syb2). Amperometric measurements of exocytosis in mouse chromaffin cells revealed that syb2 TMD mutations altered the relation between τ and Q. The effects of these mutations showed a striking periodicity, changing sign as the structural perturbation moved through the inner and outer leaflets. Some glycine and charge mutations also influenced the dependence of τ on Q in a manner consistent with expected changes in C0. These results suggest that side chains in the syb2 TMD influence the kinetics of exocytosis by perturbing the packing of the surrounding lipids. The present results support the view that membrane bending occurs during fusion pore expansion rather than during fusion pore formation. This supports the view of an initial fusion pore through two relatively flat membranes formed by protein., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Pressure affects reaction kinetics because chemical transitions involve changes in volume, and therefore pressure is a standard thermodynamic parameter to measure these volume changes. Many organisms live in environments at external pressures other than one atmosphere (0.1 MPa). Marine animals have adapted to live at depths of over 7000 m (at pressures over 70 MPa), and microorganisms living in trenches at over 110 MPa have been retrieved. Here, kinetic changes in secretion from chromaffin cells, measured as capacitance changes using the patch-clamp technique at pressures of up to 20 MPa are presented. It is known that these high pressures drastically slow down physiological functions. High hydrostatic pressure also affects the kinetics of ion channel gating and the amount of current carried by them, and it drastically slows down synaptic transmission. The results presented here indicate a similar change in volume (activation volume) of 390 ± 57 Å(3) for large dense-core vesicles undergoing fusion in chromaffin cells and for degranulation of mast cells. It is significantly larger than activation volumes of voltage-gated ion channels in chromaffin cells. This information will be useful in finding possible protein conformational changes during the reactions involved in vesicle fusion and in testing possible molecular dynamic models of secretory processes., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)