Visualization of Membrane Pore in Live Cells Reveals a Dynamic-Pore Theory Governing Fusion and Endocytosis.

Fusion is thought to open a pore to release vesicular cargoes vital for many biological processes, including exocytosis, intracellular trafficking, fertilization, and viral entry. However, fusion pores have not been observed and thus proved in live cells. Its regulatory mechanisms and functions remain poorly understood. With super-resolution STED microscopy, we observed dynamic fusion pore behaviors in live (neuroendocrine) cells, including opening, expansion, constriction, and closure, where pore size may vary between 0 and 490 nm within 26 milliseconds to seconds (vesicle size: 180-720 nm). These pore dynamics crucially determine the efficiency of vesicular cargo release and vesicle retrieval. They are generated by competition between pore expansion and constriction. Pharmacology and mutation experiments suggest that expansion and constriction are mediated by F-actin-dependent membrane tension and calcium/dynamin, respectively. These findings provide the missing live-cell evidence, proving the fusion-pore hypothesis, and establish a live-cell dynamic-pore theory accounting for fusion, fission, and their regulation.

Somatostatin modulation of initial fusion pores in Ca2+-triggered exocytosis from mouse chromaffin cells.

Somatostatin, a peptide hormone that activates G-protein-coupled receptors, inhibits the secretion of many hormones. This study investigated the mechanisms of this inhibition using amperometry recording of Ca2+-triggered catecholamine secretion from mouse chromaffin cells. Two distinct stimulation protocols, high-KCl depolarization and caffeine, were used to trigger exocytosis, and confocal fluorescence imaging was used to monitor the rise in intracellular free Ca2+. Analysis of single-vesicle fusion events (spikes) resolved the action of somatostatin on fusion pores at different stages. Somatostatin reduced spike frequency, and this reduction was accompanied by prolongation of pre-spike feet and slowing of spike rise times. This indicates that somatostatin stabilizes initial fusion pores and slows their expansion. This action on the initial fusion pore impacted the release mode to favour kiss-and-run over full-fusion. During a spike the permeability of a fusion pore peaks, declines and then settles into a plateau. Somatostatin had no effect on the plateau, suggesting no influence on late-stage fusion pores. These actions of somatostatin were indistinguishable between exocytosis triggered by high-KCl and caffeine, and fluorescence imaging showed that somatostatin had no effect on stimulus-induced rises in cytosolic Ca2+. Our findings thus demonstrate that the signalling cascades activated by somatostatin target the exocytotic machinery that controls the initial and expanding stages of fusion pores, while having no effect on late-stage fusion pores. As a result of its stronger inhibition of full-fusion compared to kiss-and-run, somatostatin will preferentially inhibit the secretion of large peptides over the secretion of small catecholamines. KEY POINTS: Somatostatin inhibits the secretion of various hormones by activating G-protein-coupled receptors. In this study, we used amperometry to investigate the mechanism by which somatostatin inhibits catecholamine release from mouse chromaffin cells. Somatostatin increased pre-spike foot lifetime and slowed fusion pore expansion. Somatostatin inhibited full-fusion more strongly than kiss-and-run. Our results suggest that the initial fusion pore is the target of somatostatin-mediated regulation of hormone release. The stronger inhibition of full-fusion by somatostatin will result in preferential inhibition of peptide release.

Deletion of the α2δ-1 calcium channel subunit increases excitability of mouse chromaffin cells.

High voltage-gated Ca2+ 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 α1 and the auxiliary ß, α2δ and γ subunits. Four genes code for the α2δ isoforms. At the mRNA level, mouse chromaffin cells (MCCs) express predominantly the CACNA2D1 gene coding for the α2δ-1 isoform. Here we show that α2δ-1 deletion led to ∼60% reduced HVCC Ca2+ influx with slower inactivation kinetics. Pharmacological dissection showed that HVCC composition remained similar in α2δ-1-/- MCCs compared to wild-type (WT), demonstrating that α2δ-1 exerts similar functional effects on all HVCC isoforms. Consistent with reduced HVCC Ca2+ influx, α2δ-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 α2δ-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 Ca2+-dependent K+ currents. Additionally, despite the reduced HVCC Ca2+ influx, the intracellular Ca2+ transients and vesicle exocytosis or endocytosis were unaltered in α2δ-1-/- MCCs compared to WT during sustained stimulation. In conclusion, our study shows that α2δ-1 genetic deletion reduces Ca2+ influx in cultured MCCs but leads to a paradoxical increase in catecholamine secretion due to increased excitability. KEY POINTS: Deletion of the α2δ-1 high voltage-gated Ca2+ channel (HVCC) subunit reduces mouse chromaffin cell (MCC) Ca2+ influx by ∼60% but causes a paradoxical increase in induced excitability. MCC intracellular Ca2+ transients are unaffected by the reduced HVCC Ca2+ influx. Deletion of α2δ-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 α2δ-1 loss-of-function mutations.

Effects of reversible SERCA inhibition on catecholamine exocytosis and intracellular [Ca2+] signaling in chromaffin cells from normotensive Wistar Kyoto rats and spontaneously hypertensive rats.

Intracellular Ca2+ ([Ca2+]i) signaling and catecholamine (CA) exocytosis from adrenal chromaffin cells (CCs) differ between mammalian species. These differences partly result from the different contributions of Ca2+-induced Ca2+-release (CICR) from internal stores, which boosts intracellular Ca2+ signals. Transient inhibition of the sarcoendoplasmic reticulum (SERCA) Ca2+ pump with cyclopiazonic acid (CPA) reduces CICR. Recently, Martínez-Ramírez et al. found that CPA had contrasting effects on catecholamine secretion and intracellular Ca2+ signals in mouse and bovine CCs, where it enhanced and inhibited exocytosis, respectively. After CPA withdrawal, exocytosis diminished in mouse CCs and increased in bovine CCs. These differences can be explained if mouse CCs have weak CICR and strong Ca2+ uptake, and the reverse is true for bovine CCs. Surprisingly, CPA slightly reduced the amplitude of Ca2+ signals in both mouse and bovine CCs. Here we examined the effects of CPA on stimulated CA exocytosis and Ca2+ signaling in rat CCs and investigated if it alters differently the responses of CCs from normotensive (WKY) or hypertensive (SHR) rats, which differ in the gain of CICR. Our results demonstrate that CPA application strongly inhibits voltage-gated exocytosis and Ca2+ transients in rat CCs, regardless of strain (SHR or WKY). Thus, despite the greater phylogenetic distance from the most recent common ancestors, suppression of endoplasmic reticulum (ER) Ca2+ uptake through CPA inhibits the CA secretion in rat CCs more similarly to bovine than mouse CCs, unveiling divergent evolutionary relationships in the mechanism of CA exocytosis of CCs between rodents. Agents that inhibit the SERCA pump, such as CPA, suppress catecholamine secretion equally well in WKY and SHR CCs and are not potential therapeutic agents for hypertension. Rat CCs display Ca2+ signals of varying widths. Some even show early and late Ca2+ components. Narrowing the Ca2+ transients by CPA and ryanodine suggests that the late component is mainly due to CICR. Simultaneous recordings of Ca2+ signaling and amperometry in CCs revealed the existence of a robust and predictable correlation between the kinetics of the whole-cell intracellular Ca2+ signal and the rate of exocytosis at the single-cell level.

The Role of Nicotinic Receptors on Ca2+ Signaling in Bovine Chromaffin Cells.

Chromaffin cells have been used as a physiological model to understand neurosecretion in mammals for many years. Nicotinic receptors located in the cells' membrane are stimulated by acetylcholine, and they participate in the exocytosis of chromaffin granules, releasing catecholamines in response to stress. In this work, we discuss how the participation of nicotinic receptors and the localization of active zones in the borders of the cytoskeleton can generate local calcium signals leading to secretion. We use a computational model of a cytoskeleton cage to simulate Ca2+ levels in response to voltage and acetylcholine pulses. We find that nicotinic receptors are able to enhance the differences between local and average calcium values, as well as the heterogeneous distributions around the active zones, producing a non-linear, highly localized Ca2+ entry that, although consisting of a few ions, is able to improve secretion responses in chromaffin cells. Our findings emphasize the intricate interplay among nicotinic receptors, the cytoskeleton, and active zones within chromaffin cells as an example of Ca2+-dependent neurosecretion in mammals.

María Teresa Miras Portugal: a pioneer in the study of purinoceptors in chromaffin cells.

María Teresa Miras Portugal devoted most of her scientific life to the study of purinergic signalling. In an important part of her work, she used a model system: the chromaffin cells of the adrenal medulla. It was in these cells that she identified diadenosine polyphosphates, from which she proceeded to the study of adrenomedullary purinome: nucleotide synthesis and degradation, adenosine transport, nucleotide uptake into chromaffin granules, exocytotic release of nucleotides and autocrine regulation of chromaffin cell function via purinoceptors. This short review will focus on the current state of knowledge of the purinoceptors of adrenal chromaffin cells, a subject to which María Teresa made seminal contributions and which she continued to study until the end of her scientific life.

Differential Modulation of Catecholamine and Adipokine Secretion by the Short Chain Fatty Acid Receptor FFAR3 and α2-Adrenergic Receptors in PC12 Cells.

Sympathetic nervous system (SNS) hyperactivity is mediated by elevated catecholamine (CA) secretion from the adrenal medulla, as well as enhanced norepinephrine (NE) release from peripheral sympathetic nerve terminals. Adrenal CA production from chromaffin cells is tightly regulated by sympatho-inhibitory α2-adrenergic (auto)receptors (ARs), which inhibit both epinephrine (Epi) and NE secretion via coupling to Gi/o proteins. α2-AR function is, in turn, regulated by G protein-coupled receptor (GPCR)-kinases (GRKs), especially GRK2, which phosphorylate and desensitize them, i.e., uncouple them from G proteins. On the other hand, the short-chain free fatty acid (SCFA) receptor (FFAR)-3, also known as GPR41, promotes NE release from sympathetic neurons via the Gi/o-derived free Gßγ-activated phospholipase C (PLC)-ß/Ca2+ signaling pathway. However, whether it exerts a similar effect in adrenal chromaffin cells is not known at present. In the present study, we examined the interplay of the sympatho-inhibitory α2A-AR and the sympatho-stimulatory FFAR3 in the regulation of CA secretion from rat adrenal chromaffin (pheochromocytoma) PC12 cells. We show that FFAR3 promotes CA secretion, similarly to what GRK2-dependent α2A-AR desensitization does. In addition, FFAR3 activation enhances the effect of the physiologic stimulus (acetylcholine) on CA secretion. Importantly, GRK2 blockade to restore α2A-AR function or the ketone body beta-hydroxybutyrate (BHB or 3-hydroxybutyrate), via FFAR3 antagonism, partially suppress CA production, when applied individually. When combined, however, CA secretion from PC12 cells is profoundly suppressed. Finally, propionate-activated FFAR3 induces leptin and adiponectin secretion from PC12 cells, two important adipokines known to be involved in tissue inflammation, and this effect of FFAR3 is fully blocked by the ketone BHB. In conclusion, SCFAs can promote CA and adipokine secretion from adrenal chromaffin cells via FFAR3 activation, but the metabolite/ketone body BHB can effectively inhibit this action.

Biogenesis of large dense core vesicles in mouse chromaffin cells.

Large dense core vesicle (LDCVs) biogenesis in neuroendocrine cells involves: (a) production of cargo peptides processed in the Golgi; (b) fission of cargo loaded LDCVs undergoing maturation steps; (c) movement of these LDCVs to the plasma membrane. These steps have been resolved over several decades in PC12 cells and in bovine chromaffin cells. More recently, the molecular machinery involved in LDCV biogenesis has been examined using genetically modified mice, generating contradictory results. To address these contradictions, we have used NPY-mCherry electroporation combined with immunolabeling and super-resolution structured illumination microscopy. We show that LDCVs separate from an intermediate Golgi compartment, mature in its proximity for about 1 hour and then travel to the plasma membrane. The exocytotic machinery composed of vSNAREs and synaptotagmin1, which originate from either de novo synthesis or recycling, is most likely acquired via fusion with precursor vesicles during maturation. Finally, recycling of LDCV membrane protein is achieved in less than 2 hours. With this comprehensive scheme of LDCV biogenesis we have established a framework for future studies in mouse chromaffin cells.

Cross Talk between α7 and α3ß4 Nicotinic Receptors Prevents Their Desensitization in Human Chromaffin Cells.

The physical interaction and functional cross talk among the different subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) expressed in the various tissues is unknown. Here, we have investigated this issue between the only two nAChRs subtypes expressed, the α7 and α3ß4 subtypes, in a human native neuroendocrine cell (the chromaffin cell) using electrophysiological patch-clamp, fluorescence, and Förster resonance energy transfer (FRET) techniques. Our data show that α7 and α3ß4 receptor subtypes require their mutual and maximal efficacy of activation to increase their expression, to avoid their desensitization, and therefore, to increase their activity. In this way, after repetitive stimulation with acetylcholine (ACh), α7 and α3ß4 receptor subtypes do not desensitize, but they do with choline. The nicotinic current increase associated with the α3ß4 subtype is dependent on Ca2+ In addition, both receptor subtypes physically interact. Interaction and expression of both subtypes are reversibly reduced by tyrosine and serine/threonine phosphatases inhibition, not by Ca2+ In addition, expression is greater in human chromaffin cells from men compared to women, but FRET efficiency is not affected. Together, our findings indicate that human α7 and α3ß4 subtypes mutually modulate their expression and activity, providing a promising line of research to pharmacologically regulate their activity.SIGNIFICANCE STATEMENT Desensitization of nicotinic receptors is accepted to occur with repetitive agonist stimulation. However, here we show that human native α3ß4 and α7 nicotinic acetylcholine receptor (nAChR) subtypes do not desensitize, and instead, increase their activity when they are activated by the physiological agonist acetylcholine (ACh). An indispensable requirement is the activation of the other receptor subtype with maximal efficacy, and the presence of Ca2+ to cooperate in the case of the α3ß4 current increase. Because choline is an α3ß4 partial agonist, it will act as a limiting factor of nicotinic currents enhancement in the absence of ACh, but in its presence, it will further potentiate α7 currents.

CLC Anion/Proton Exchangers Regulate Secretory Vesicle Filling and Granule Exocytosis in Chromaffin Cells.

ClC-3, ClC-4, and ClC-5 are electrogenic chloride/proton exchangers that can be found in endosomal compartments of mammalian cells. Although the association with genetic diseases and the severe phenotype of knock-out animals illustrate their physiological importance, the cellular functions of these proteins have remained insufficiently understood. We here study the role of two Clcn3 splice variants, ClC-3b and ClC-3c, in granular exocytosis and catecholamine accumulation of adrenal chromaffin cells using a combination of high-resolution capacitance measurements, amperometry, protein expression/gene knock out/down, rescue experiments, and confocal microscopy. We demonstrate that ClC-3c resides in immature as well as in mature secretory granules, where it regulates catecholamine accumulation and contributes to the establishment of the readily releasable pool of secretory vesicles. The lysosomal splice variant ClC-3b contributes to vesicle priming only with low efficiency and leaves the vesicular catecholamine content unaltered. The related Cl-/H+ antiporter ClC-5 undergoes age-dependent downregulation in wild-type conditions. Its upregulation in Clcn3-/- cells partially rescues the exocytotic mutant defect. Our study demonstrates how different CLC transporters with similar transport functions, but distinct localizations can contribute to vesicle functions in the regulated secretory pathway of granule secretion in chromaffin cells.SIGNIFICANCE STATEMENT Cl-/H+ exchangers are expressed along the endosomal/lysosomal system of mammalian cells; however, their exact subcellular functions have remained insufficiently understood. We used chromaffin cells, a system extensively used to understand presynaptic mechanisms of synaptic transmission, to define the role of CLC exchangers in neurosecretion. Disruption of ClC-3 impairs catecholamine accumulation and secretory vesicle priming. There are multiple ClC-3 splice variants, and only expression of one, ClC-3c, in double Cl-/H+ exchanger-deficient cells fully rescues the WT phenotype. Another splice variant, ClC-3b, is present in lysosomes and is not necessary for catecholamine secretion. The distinct functions of ClC-3c and ClC-3b illustrate the impact of expressing multiple CLC transporters with similar transport functions and separate localizations in different endosomal compartments.

Forty years of the adrenal chromaffin cell through ISCCB meetings around the world.

This historical review focuses on the evolution of the knowledge accumulated during the last two centuries on the biology of the adrenal medulla gland and its chromaffin cells (CCs). The review emerged in the context of a series of meetings that started on the Spanish island of Ibiza in 1982 with the name of the International Symposium on Chromaffin Cell Biology (ISCCB). Hence, the review is divided into two periods namely, before 1982 and from this year to 2022, when the 21st ISCCB meeting was just held in Hamburg, Germany. The first historical period extends back to 1852 when Albert Kölliker first described the fine structure and function of the adrenal medulla. Subsequently, the adrenal staining with chromate salts identified the CCs; this was followed by the establishment of the embryological origin of the adrenal medulla, and the identification of adrenaline-storing vesicles. By the end of the nineteenth century, the basic morphology, histochemistry, and embryology of the adrenal gland were known. The twentieth century began with breakthrough findings namely, the experiment of Elliott suggesting that adrenaline was the sympathetic neurotransmitter, the isolation of pure adrenaline, and the deciphering of its molecular structure and chemical synthesis in the laboratory. In the 1950s, Blaschko isolated the catecholamine-storing vesicles from adrenal medullary extracts. This switched the interest in CCs as models of sympathetic neurons with an explosion of studies concerning their functions, i.e., uptake of catecholamines by chromaffin vesicles through a specific coupled transport system; the identification of several vesicle components in addition to catecholamines including chromogranins, ATP, opioids, and other neuropeptides; the calcium-dependence of the release of catecholamines; the underlying mechanism of exocytosis of this release, as indicated by the co-release of proteins; the cross-talk between the adrenal cortex and the medulla; and the emission of neurite-like processes by CCs in culture, among other numerous findings. The 1980s began with the introduction of new high-resolution techniques such as patch-clamp, calcium probes, marine toxins-targeting ion channels and receptors, confocal microscopy, or amperometry. In this frame of technological advances at the Ibiza ISCCB meeting in 1982, 11 senior researchers in the field predicted a notable increase in our knowledge in the field of CCs and the adrenal medulla; this cumulative knowledge that occurred in the last 40 years of history of the CC is succinctly described in the second part of this historical review. It deals with cell excitability, ion channel currents, the exocytotic fusion pore, the handling of calcium ions by CCs, the kinetics of exocytosis and endocytosis, the exocytotic machinery, and the life cycle of secretory vesicles. These concepts together with studies on the dynamics of membrane fusion with super-resolution imaging techniques at the single-protein level were extensively reviewed by top scientists in the field at the 21st ISCCB meeting in Hamburg in the summer of 2022; this frontier topic is also briefly reviewed here. Many of the concepts arising from those studies contributed to our present understanding of synaptic transmission. This has been studied in physiological or pathophysiological conditions, in CCs from animal disease models. In conclusion, the lessons we have learned from CC biology as a peripheral model for brain and brain disease pertain more than ever to cutting-edge research in neurobiology. In the 22nd ISCCB meeting in Israel in 2024 that Uri Asheri is organizing, we will have the opportunity of seeing the progress of the questions posed in Ibiza, and on other questions that undoubtedly will arise.

Aluminum alters excitability by inhibiting calcium, sodium, and potassium currents in bovine chromaffin cells.

Aluminum (Al3+ ) has long been related to neurotoxicity and neurological diseases. This study aims to describe the specific actions of this metal on cellular excitability and neurotransmitter release in primary culture of bovine chromaffin cells. Using voltage-clamp and current-clamp recordings with the whole-cell configuration of the patch clamp technique, online measurement of catecholamine release, and measurements of [Ca2+ ]c with Fluo-4-AM, we have observed that Al3+ reduced intracellular calcium concentrations around 25% and decreased catecholamine secretion in a dose-dependent manner, with an IC50 of 89.1 µM. Al3+ blocked calcium currents in a time- and concentration-dependent manner with an IC50 of 560 µM. This blockade was irreversible since it did not recover after washout. Moreover, Al3+ produced a bigger blockade on N-, P-, and Q-type calcium channels subtypes (69.5%) than on L-type channels subtypes (50.5%). Sodium currents were also inhibited by Al3+ in a time- and concentration-dependent manner, 24.3% blockade at the closest concentration to the IC50 (399 µM). This inhibition was reversible. Voltage-dependent potassium currents were low affected by Al3+ . Nonetheless, calcium/voltage-dependent potassium currents were inhibited in a concentration-dependent manner, with an IC50 of 447 µM. This inhibition was related to the depression of calcium influx through voltage-dependent calcium channels subtypes coupled to BK channels. In summary, the blockade of these ionic conductance altered cellular excitability that reduced the action potentials firing and so, the neurotransmitter release and the synaptic transmission. These findings prove that aluminum has neurotoxic properties because it alters neuronal excitability by inhibiting the sodium currents responsible for the generation and propagation of impulse nerve, the potassium current responsible for the termination of action potentials, and the calcium current responsible for the neurotransmitters release.

Mitochondrial dysfunction in chromaffin cells from the R6/1 mouse model of Huntington's disease: Impact on exocytosis and calcium current regulation.

From a pathogenic perspective, Huntington's disease (HD) is being considered as a synaptopathy. As such, alterations in brain neurotransmitter release occur. As the activity of the sympathoadrenal axis is centrally controlled, deficits in the exocytotic release of catecholamine release may also occur. In fact, in chromaffin cells (CCs) of the adrenal medulla of the R6/1 model of HD, decrease of secretion and altered kinetics of the exocytotic fusion pore have been reported. Those alterations could be linked to mitochondrial deficits occurring in peripheral CCs, similar to those described in brain mitochondria. Here we have inquired about alterations in mitochondrial structure and function and their impact on exocytosis and calcium channel currents (ICa). We have monitored various parameters linked to those events, in wild type (WT) and the R6/1 mouse model of HD at a pre-disease stage (2 months age, 2 m), and when motor deficits are present (7 months age, 7 m). In isolated CCs from 7 m and in the adrenal medulla of R6/1 mice, we found the following alterations (with respect 7 m WT mice): (i) augmented fragmented mitochondria and oxidative stress with increased oxidized glutathione; (ii) decreased basal and maximal respiration; (iii) diminution of ATP cell levels; (iv) mitochondrial depolarization; (v) drastic decrease of catecholamine release with poorer potentiation by protonophore FCCP; (vi) decreased ICa inhibition by FCCP; and (vii) lesser potentiation by BayK8644 of ICa and smaller prolongation of current deactivation. Of note was the fact several of these alterations were already manifested in CCs from 2 m R6/1 mice at pre-disease stages. Based on those results, a plausible hypothesis can be raised in the sense that altered mitochondrial function seems to be an early primary event in HD pathogenesis. This is in line with an increasing number of mitochondrial, metabolic, and inflammatory alterations being recently reported in various HD peripheral tissues.

Expression and function of mitochondrial inhibitor factor-1 and TASK channels in adrenal cells.

Adrenal medullary chromaffin (AMC) cells in the perinatal period and carotid body glomus cells after birth respond to hypoxia with catecholamine secretion. The hypoxia detection mechanism in such O2-sensitive cells is still not well defined. One hypothesis is that a decrease in cellular ATP may be involved in the hypoxia detection. This idea is based on ATP dependence of TASK channel activity that regulates the resting membrane potential and is suppressed by hypoxia in glomus cells. Mitochondrial ATPase inhibitor factor-1 (IF1), a physiological regulator of ATP synthase, helps prevent ATP hydrolysis under hypoxic conditions. In cells where IF1 expression is high, exposure to hypoxia is expected to have no effect on TASK channel activity. This possibility was electrophysiologically and immunocytochemically explored. Single channel recordings revealed that 36-pS TASK3-like channels contribute to the resting membrane potential in young rat adrenal cortical (AC) cells. TASK3-like channel activity in a cell-attached patch was not affected by bath application of mitochondrial inhibitors. Consistent with this finding, IF1-like immunoreactive material was well expressed in rat AC cells. In further support of our hypothesis, IF1-like immunoreactive material was well expressed in adult rat AMC cells that are known to be hypoxia-insensitive and minimally expressed in newborn AMC cells that are hypoxia-sensitive. These results provide evidence for the functional relevance of IF1 expression in excitability in O2-sensitive cells in response to mitochondrial inhibition.

Regulating quantal size of neurotransmitter release through a GPCR voltage sensor.

Current models emphasize that membrane voltage (Vm) depolarization-induced Ca2+ 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 Gißγ subunits with exocytosis fusion proteins. Here we report that, independently from Ca2+, 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-P2Y12 receptors. D76 and D127 in P2Y12 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 → P2Y12(D76/D127) → Gißγ → QS → myocyte contractility, offering a universal Vm-GPCR signaling pathway for its functions in the nervous system and other systems containing GPCRs.

Regulation of Morphogenetic Processes during Postnatal Development and Physiological Regeneration of the Adrenal Medulla.

Regulation of morphogenetic processes during postnatal development of the rat adrenal medulla was studied. Termination of the adrenal medulla growth was found to be associated with decreased chromaffin cell proliferation, activation of canonical Wnt-signaling pathway, and enhanced expression of Sonic Hedgehog ligand. Analysis of transcription factors associated with pluripotency revealed increased percentage of Oct4-expressing cells by the end of medulla growth and no signs of Sox2 expression. All the cells demonstrating activation of Wnt-signaling and expression of Oct4 and Sonic Hedgehog were found to be highly differentiated chromaffin cells actively producing tyrosine hydroxylase. These findings allow considering the formation of the cell pools for dedifferentiation as a putative mechanism for physiological regeneration of the adrenal medulla.

MASH1 induces neuron transdifferentiation of adrenal medulla chromaffin cells. / MASH1ä»‹å¯¼è‚¾ä¸Šè ºé«“è´¨å—œé“¬ç»†èƒžå‘ç”Ÿç¥žç»å ƒè½¬åˆ†åŒ–.

OBJECTIVES: Nerve growth factor (NGF) induces neuron transdifferentiation of adrenal medulla chromaffin cells (AMCCs) and consequently downregulates the secretion of epinephrine (EPI), which may be involved in the pathogenesis of bronchial asthma. Mammalian achaete scute-homologous 1 (MASH1), a key regulator of neurogenesis in the nervous system, has been proved to be elevated in AMCCs with neuron transdifferentiation in vivo. This study aims to explore the role of MASH1 in the process of neuron transdifferentiation of AMCCs and the mechanisms. METHODS: Rat AMCCs were isolated and cultured. AMCCs were transfected with siMASH1 or MASH1 overexpression plasmid, then were stimulated with NGF and/or dexamethasone, PD98059 (a MAPK kinase-1 inhibitor) for 48 hours. Morphological changes were observed using light and electron microscope. Phenylethanolamine-N-methyltransferase (PNMT, the key enzyme for epinephrine synthesis) and tyrosine hydroxylase were detected by immunofluorescence. Western blotting was used to test the protein levels of PNMT, MASH1, peripherin (neuronal markers), extracellular regulated protein kinases (ERK), phosphorylated extracellular regulated protein kinases (pERK), and JMJD3. Real-time RT-PCR was applied to analyze the mRNA levels of MASH1 and JMJD3. EPI levels in the cellular supernatant were measured using ELISA. RESULTS: Cells with both tyrosine hydroxylase and PNMT positive by immunofluorescence were proved to be AMCCs. Exposure to NGF, AMCCs exhibited neurite-like processes concomitant with increases in pERK/ERK, peripherin, and MASH1 levels (all P<0.05). Additionally, impairment of endocrine phenotype was proved by a signifcant decrease in the PNMT level and the secretion of EPI from AMCCs (all P<0.01). MASH1 interference reversed the effect of NGF, causing increases in the levels of PNMT and EPI, conversely reduced the peripherin level and cell processes (all P<0.01). MASH1 overexpression significantly increased the number of cell processes and peripherin level, while decreased the levels of PNMT and EPI (all P<0.01). Compared with the NGF group, the levels of MASH1, JMJD3 protein and mRNA in AMCCs in the NGF+PD98059 group were decreased (all P<0.05). After treatment with PD98059 and dexamethasone, the effect of NGF on promoting the transdifferentiation of AMCCs was inhibited, and the number of cell processes and EPI levels were decreased (both P<0.05). In addition, the activity of the pERK/MASH1 pathway activated by NGF was also inhibited. CONCLUSIONS: MASH1 is the key factor in neuron transdifferentiation of AMCCs. NGF-induced neuron transdifferentiation is probably mediated via pERK/MASH1 signaling.

Synaptophysin Regulates Fusion Pores and Exocytosis Mode in Chromaffin Cells.

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 Ca2+-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 Ca2+-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.

Ultrashort nanosecond electric pulses activate a conductance in bovine adrenal chromaffin cells that involves cation entry through TRPC and NALCN channels.

In whole-cell voltage clamped bovine adrenal chromaffin cells maintained at a holding potential of -70 mV, a single 5 ns, 5 MV/m pulse elicited an inward current carried mainly by Na+ that displayed inward rectification and a reversal potential near -3 mV, a voltage consistent with a non-selective cation current. The broad-spectrum inhibitors of transient receptor potential (TRP) channels, La3+ (10 µM), Gd3+ (10 µM), SKF-96365 (50 µM) and 2-aminoethoxydiphenyl borane (2-APB; 100 µM), inhibited the current similarly by ∼72%, ∼83%, ∼68% and ∼76%, respectively. Depleting membrane cholesterol with methyl-ß-cyclodextrin (MßCD; 1-6 mg/ml) or inhibiting phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis with wortmannin (20 and 40 µM) produced a similar level of inhibition on the NEP-induced conductance as the broad spectrum TRP channel inhibitors. Moreover, no additive inhibitory effect was detected by combining MßCD (3 mg/ml), wortmannin (20 µM) and La3+ (10 µM), suggesting that each agent targeted different levels of the same pathway to exert a full effect. RT-PCR experiments revealed robust expression at the mRNA level of TRPC4, TRPC5 and TRPM7 channels for which specific blockers were available. Whereas the TRPM7 blocker FTY720 had no effect, the TRPC4/5 channel inhibitor M084 (20 µM) blocked the conductance by ∼50%, indicating that TRPC4 and/or TRPC5 channel(s) may be partially involved in mediating the NEP-induced current. CP-96345 (20 µM), a specific blocker of the sodium leak current channel (NALCN), also reduced the NEP-induced current. The inhibition was ∼30% and additive to that caused by the TRPC4/5 blocker M084. RT-PCR experiments confirmed the expression of this channel at the mRNA level. Taken as a whole, these data provide evidence that a large fraction of the current evoked by a 5 ns pulse in adrenal chromaffin cells may be carried by both TRPC4/5 channels and the NALCN channel. Understanding the biophysical properties of the NEP-elicited conductance in a neural-type cell will be extremely valuable for the future development of NEP stimulation approaches for neuromodulation.

María Teresa Miras Portugal: a pioneer for vesicular nucleotide storage.

Chromaffin granules are secretory granules present in adrenal medulla chromaffin cells. They contain high contents of catecholamines and nucleotides and have been regarded as a model system for the study of vesicular transmitter uptake and release. In 1988, Dr. María Teresa Miras Portugal, when studying catecholamine biosynthesis, detected a novel group of nucleotides, the diadenosine polyphosphates, in the adrenal chromaffin granules. Based on this finding, she unraveled the existence of diadenosine polyphosphate-mediated chemical transmission, leading to a paradigm shift in the field of purinergic signaling. She is also a pioneer in the studies on vesicular nucleotide storage. First, María Teresa and her group characterized nucleotide transport in chromaffin granules and synaptic vesicles using fluorescent nucleotide derivatives such as 1, N6-ethenoadenosine triphosphates. Then, they revealed the presence of a hypothetical vesicular nucleotide transporter with unique properties in terms of substrate specificity. In this article, we will describe her contributions to vesicular nucleotide storage and the foundations she laid for future studies.