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Key points 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. 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. [ABSTRACT FROM AUTHOR]

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 β, 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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Phox2B is a transcription factor responsible for chromaffin cell phenotype. Although it is used routinely for diagnosis of neuroblastoma, previous reports concerning its utility in the diagnosis of neuroendocrine neoplasms have been conflicting. We assessed Phox2b immunoreactivity in different neuroendocrine neoplasms. Tissue microarrays or whole sections of 36 paragangliomas (PGs), 91 well-differentiated neuroendocrine tumours of different organs (WDNETs), 31 neuroendocrine carcinomas (NECs), and 6 olfactory neuroblastomas (ONBs) were stained with Phox2B antibody (EP312) and GATA3. The percentage of positive cells and intensity was analysed using H-score. Phox2B immunoreactivity was seen in 97.2% (35/36) PGs, 11% (10/91) WDNETs, 9.7% (3/31) NECs, and 16.7% (1/6) ONBs. PGs were significantly more often positive (p < 0.001, χ2) than other neuroendocrine tumours, showing highest H-score (mean 144.9, SD ± 75.1) and percentage of positive cells (median 81.3%, IQR 62.5–92.5%). Compared to Phox2B-positive WDNETs, PGs showed significantly higher H-score (median 145 vs 7.5, p < 0.001) and percentage of positive cells (median 82.5% vs 4.5%, p < 0.001). Phox2B positivity was 97.2% sensitive and 89% specific for the diagnosis of PG. GATA3 was 100% sensitive and 88% specific for the diagnosis of PG. When combined, any Phox2B/GATA3 coexpression was 97.1% sensitive and 99.1% specific for the diagnosis of paraganglioma. Widespread Phox2B immunoreactivity is a highly characteristic feature of PGs and it can be used as an additional marker in differential diagnosis of neuroendocrine tumours. [ABSTRACT FROM AUTHOR]

The cortical actin network is a mesh of filaments distributed beneath the plasmalemma that dynamically reacts in response to stimuli. This dynamic network of cortical filaments, together with motor myosin partners, adjusts the plasmalemma tension, organizes membrane protein microdomains, remodels the cell surface and drives vesicle motion in order to fine-tune exocytosis, endocytosis and recycling of secretory vesicles. In this review, we discuss how these mechanisms work in secretory cells. [ABSTRACT FROM AUTHOR]

During embryonic development, nerve-associated Schwann cell precursors (SCPs) give rise to chromaffin cells of the adrenal gland via the "bridge" transient stage, according to recent functional experiments and single cell data from humans and mice. However, currently existing data do not resolve the finest heterogeneity of developing chromaffin populations. Here we took advantage of deep SmartSeq2 transcriptomic sequencing to expand our collection of individual cells from the developing murine sympatho-adrenal anlage and uncover the microheterogeneity of embryonic chromaffin cells and their corresponding developmental paths. We discovered that SCPs on the splachnic nerve show a high degree of microheterogeneity corresponding to early biases towards either Schwann or chromaffin terminal fates. Furthermore, we found that a post-"bridge" population of developing chromaffin cells gives rise to persisting oxygen-sensing chromaffin cells and the two terminal populations (adrenergic and noradrenergic) via diverging differentiation paths. Taken together, we provide a thorough identification of novel markers of adrenergic and noradrenergic populations in developing adrenal glands and report novel differentiation paths leading to them. [ABSTRACT FROM AUTHOR]

During embryonic development, nerve-associated Schwann cell precursors (SCPs) give rise to chromaffin cells of the adrenal gland via the “bridge” transient stage, according to recent functional experiments and single cell data from humans and mice. However, currently existing data do not resolve the finest heterogeneity of developing chromaffin populations. Here we took advantage of deep SmartSeq2 transcriptomic sequencing to expand our collection of individual cells from the developing murine sympatho-adrenal anlage and uncover the microheterogeneity of embryonic chromaffin cells and their corresponding developmental paths. We discovered that SCPs on the splachnic nerve show a high degree of microheterogeneity corresponding to early biases towards either Schwann or chromaffin terminal fates. Furthermore, we found that a post-”bridge” population of developing chromaffin cells gives rise to persisting oxygen-sensing chromaffin cells and the two terminal populations (adrenergic and noradrenergic) via diverging differentiation paths. Taken together, we provide a thorough identification of novel markers of adrenergic and noradrenergic populations in developing adrenal glands and report novel differentiation paths leading to them.

The adrenal medulla plays a critical role in mammalian homeostasis and the stress response. It is populated by clustered chromaffin cells that secrete epinephrine or norepinephrine along with peptides into the bloodstream affecting distant target organs. Despite been heavily studied, the central control of adrenal medulla and in-situ spatiotemporal responsiveness remains poorly understood. For this work, we continuously monitored the electrical activity of individual adrenomedullary chromaffin cells in the living anesthetized rat using multielectrode arrays. We measured the chromaffin cell activity under basal and physiological stress conditions and characterized the functional micro-architecture of the adrenal medulla. Under basal conditions, chromaffin cells fired action potentials with frequencies between ~0.2 and 4 Hz. Activity was almost completely driven by sympathetic inputs coming through the splanchnic nerve. Chromaffin cells were organized into independent local networks in which cells fired in a specific order, with latencies from hundreds of microseconds to a few milliseconds. Electrical stimulation of the splanchnic nerve evoked almost exactly the same spatiotemporal firing patterns that occurred spontaneously. Hypoglycemic stress, induced by insulin administration resulted in increased activity of a subset of the chromaffin cells. In contrast, respiratory arrest induced by lethal anesthesia resulted in an increase in the activity of virtually all chromaffin cells before cessation of all activity. These results suggest a stressor-specific activation of adrenomedullary chromaffin cell networks and revealed a surprisingly complex electrical organization that likely reflects the dynamic nature of the adrenal medulla's neuroendocrine output during basal conditions and during different types of physiological stress. [ABSTRACT FROM AUTHOR]

Simple Summary: Neuroblastoma is a childhood tumor of the sympathetic nervous system. Abdominal tumors that arise in the adrenal differ genetically and clinically from tumors that develop from sympathetic ganglia. Adrenal chromaffin cells and ganglionic neuroblasts are derived from different lineages, raising the possibility that their diverse tumor characteristics are due to different tumor founder cells. To identify traits that are specific to the candidate founder cells of adrenal and ganglionic tumors, cultures of mouse chromaffin cells and sympathetic neuroblasts were treated with a panel of proliferation inhibitors that affect various signaling pathways. Transcription-related inhibitors (BET, CDK7/12/13) showed differential effects, indicating that the BET protein and CDK signaling pathways differ in their relevance for proliferating chromaffin cells and sympathetic neuroblasts. Thus, the oncogenic aberrations affecting these pathways should differ in their efficiency and result in selective propagation, which may lead to adrenal and ganglionic tumors having different characteristics. Neuroblastoma arising from the adrenal differ from ganglionic neuroblastoma both genetically and clinically, with adrenal tumors being associated with a more severe prognosis. The different tumor properties may be linked to specific tumor founder cells in adrenal and sympathetic ganglia. To address this question, we first set up cultures of mouse sympathetic neuroblasts and adrenal chromaffin cells. These cultures were then treated with various proliferation inhibitors to identify lineage-specific responses. We show that neuroblast and chromaffin cell proliferation was affected by WNT, ALK, IGF1, and PRC2/EZH2 signaling inhibitors to a similar extent. However, differential effects were observed in response to bromodomain and extraterminal (BET) protein inhibitors (JQ1, GSK1324726A) and to the CDK-7 inhibitor THZ1, with BET inhibitors preferentially affecting chromaffin cells, and THZ1 preferentially affecting neuroblasts. The differential dependence of chromaffin cells and neuroblasts on BET and CDK signaling may indicate different mechanisms during tumor initiation in sympathetic ganglia and adrenal. [ABSTRACT FROM AUTHOR]

Secretogranin II (SgII) and III (SgIII) function within peptide hormone-producing cells and are involved in secretory granule formation. However, their function in active amine-producing cells is not fully understood. In this study, we analyzed the expression profiles of SgII and SgIII in canine adrenal medulla and pheochromocytomas by immunohistochemical staining. In normal adrenal tissues, the intensity of coexpression of these two secretogranins (Sgs) differed from each chromaffin cell, although a complete match was not observed. The coexpression of vesicular monoamine transporter 2 (VMAT2) with SgIII was similar to that with chromogranin A, but there was a subpopulation of VMAT2-expressing cells that were negative or hardly detectable for SgII. These results are the first to indicate that there are distinct expression patterns for SgII and SgIII in adrenal chromaffin cells. Furthermore, the expression of these two Sgs varied in intensity among pheochromocytomas and did not necessarily correlate with clinical plasma catecholamine levels in patients. However, compared with SgIII, the expression of SgII was shown to be strong at the single-cell level in some tumor tissues. These findings provide a fundamental understanding of the expression differences between SgII and SgIII in normal adrenal chromaffin cells and pheochromocytomas. [ABSTRACT FROM AUTHOR]

Simple Summary: Alzheimer's disease (AD), the most common form of dementia, is becoming a global health problem and public health priority. In the advanced stages of AD, besides the initial cognitive symptoms, behavioral problems, particularly agitation and aggressiveness, become prevalent in AD patients. These non-cognitive symptoms could be related to alterations in the regulatory mechanism of the sympathetic nervous system. In this study, we used chromaffin cells (CCs) isolated from the adrenal gland of 3xTg (an AD mouse model) mice to characterize potential alterations in the regulation of the responses to stress mediated by the secretion of catecholamines. We compared these regulatory mechanisms in mice at two different ages: in 2-month-old mice, where no AD symptoms were observed, and in mice over 12 months of age, when AD-related cognitive impairment related was fully established. We found that the modulation of neurotransmitter release was stronger in CCs isolated from the adrenal medulla of 3xTg mice older than 12 months of age, an effect likely related to disease progression as it was not observed in CCs from age-matched wild-type (WT) mice. This enhanced modulation leads to an increased catecholamine release in response to stressful situations, which may explain the non-cognitive behavioral problems found in AD patients. Alzheimer's disease (AD), the most common form of dementia, is becoming a global health problem and public health priority. In the advanced stages of AD, besides the initial cognitive symptoms, behavioral problems, particularly agitation and aggressiveness, become prevalent in AD patients. These non-cognitive symptoms could be related to a noradrenergic overactivation. In this study, we used chromaffin cells (CCs) isolated from the adrenal gland of 3xTg AD model mice to characterize potential alterations in the autocrine-paracrine modulation of voltage-dependent calcium channels (VDCCs), which in turn serve to regulate the release of catecholamines. We used mice at the presymptomatic stage (2 months) and mice over 12 months of age, when AD-related cognitive impairment was fully established. We found that the modulation of inward currents through VDCCs induced by extracellular ATP was stronger in CCs isolated from the adrenal medulla of 3xTg mice older than 12 months of age, an effect likely related to disease progression as it was not observed in CCs from age-matched WT mice. This enhanced modulation leads to increased catecholamine release in response to stressful situations, which may explain the non-cognitive behavioral problems found in AD patients. [ABSTRACT FROM AUTHOR]

The adrenal medulla plays a critical role in mammalian homeostasis and the stress response. It is populated by clustered chromaffin cells that secrete epinephrine or norepinephrine along with peptides into the bloodstream affecting distant target organs. Despite been heavily studied, the central control of adrenal medulla and in-situ spatiotemporal responsiveness remains poorly understood. For this work, we continuously monitored the electrical activity of individual adrenomedullary chromaffin cells in the living anesthetized rat using multielectrode arrays. We measured the chromaffin cell activity under basal and physiological stress conditions and characterized the functional micro-architecture of the adrenal medulla. Under basal conditions, chromaffin cells fired action potentials with frequencies between ~0.2 and 4 Hz. Activity was almost completely driven by sympathetic inputs coming through the splanchnic nerve. Chromaffin cells were organized into independent local networks in which cells fired in a specific order, with latencies from hundreds of microseconds to a few milliseconds. Electrical stimulation of the splanchnic nerve evoked almost exactly the same spatiotemporal firing patterns that occurred spontaneously. Hypoglycemic stress, induced by insulin administration resulted in increased activity of a subset of the chromaffin cells. In contrast, respiratory arrest induced by lethal anesthesia resulted in an increase in the activity of virtually all chromaffin cells before cessation of all activity. These results suggest a stressor-specific activation of adrenomedullary chromaffin cell networks and revealed a surprisingly complex electrical organization that likely reflects the dynamic nature of the adrenal medulla’s neuroendocrine output during basal conditions and during different types of physiological stress.

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 α3β4 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 α3β4 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 α3β4 receptor subtypes do not desensitize, but they do with choline. The nicotinic current increase associated with the α3β4 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 α3β4 subtypes mutually modulate their expression and activity, providing a promising line of research to pharmacologically regulate their activity. [ABSTRACT FROM AUTHOR]

The hypersecretory phenotype of adrenal chromaffin cells (CCs) from early spontaneously hypertensive rats (SHRs) mainly results from enhanced Ca2+-induced Ca2+-release (CICR). A key question is if these abnormalities can be traced to the prehypertensive stage. Spontaneous and stimulus-induced catecholamine exocytosis, intracellular Ca2+ 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 [Ca2+]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 Ca2+ 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. [ABSTRACT FROM AUTHOR]

Synaptotagmins confer calcium-dependence to the exocytosis of secretory vesicles, but how coexpressed synaptotagmins interact remains unclear. We find that synaptotagmin-1 and synaptotagmin-7 when present alone act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-1 and synaptotagmin-7 are found in largely non-overlapping clusters on dense-core vesicles. Synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming, and it promotes ubMunc13-2- and phorbolester-dependent priming, especially at low resting calcium concentrations. The priming effect of synaptotagmin-7 increases the number of vesicles fusing via synaptotagmin-1, while negatively affecting their fusion speed, indicating both synergistic and competitive interactions between synaptotagmins. Synaptotagmin-7 places vesicles in close membrane apposition (

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. [ABSTRACT FROM AUTHOR]

Glutamate and its receptors have been demonstrated to promote both basal and nicotine-evoked catecholamine release in bovine chromaffin cells. Multiple glutamate receptors, including metabotropic glutamate receptors (mGluRs), are found in the adrenal glands of several species, as well as in chromaffin cells. However, there is limited information available regarding the expression of glutamate metabotropic receptor (GRM)1-8 mRNAs and the detailed localization of group I mGluRs (mGluR1 and mGluR5) in the rat and human adrenal cortex and medulla. Therefore, we examined mRNA expression of GRM1-8 subunits using reverse transcription-polymerase chain reaction (RT-PCR) and the distribution of mGluR1 and mGluR5 by immunostaining. The results showed that the GRM1-8 mRNAs were expressed in both the cortex and medulla of rat and human adrenal glands with the exception of GRM1, which was not detectable in the rat adrenal cortex. Immunostaining of mGluR1 revealed that it was localized only in the adrenal medulla of rats but was present in both the adrenal cortex and medulla in humans. In the adrenal medulla, the central part of the adrenal glands, mGluR1 was detected in chromaffin cells but not in nerve fibers and ganglion cells. Immunoactivity of mGluR5 was visible in the capillary wall throughout the adrenal cortex and medulla in rat and human samples. Its immunoactivity was also observed in ganglion cells in the rat adrenal medulla. There was no mGluR5 immunoactivity detected in chromaffin cells and nerve fibers in the rat and human adrenal medulla. Using dissected rat adrenal medulla as a model, we found that treatment with a mGluR1 agonist activated extracellular signal-regulated kinase (ERK) 1/2 and increased the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis. Moreover, these results showed that mGluR1 signaling was involved in hypoxia-induced upregulation of TH in the rat adrenal medulla. This study shows the expression of GRM1-8 mRNAs in rat and human adrenal glands and indicates that glutamate, through the activation of mGluRs, may play various physiological roles in the adrenal gland. Furthermore, mGluR1 may be involved in catecholamine biosynthesis by regulating TH, and mGluR5 may affect cortical and medullar hormone levels by regulating microvascular function.

Alzheimer’s disease (AD), the most common form of dementia, is becoming a global health problem and public health priority. In the advanced stages of AD, besides the initial cognitive symptoms, behavioral problems, particularly agitation and aggressiveness, become prevalent in AD patients. These non-cognitive symptoms could be related to a noradrenergic overactivation. In this study, we used chromaffin cells (CCs) isolated from the adrenal gland of 3xTg AD model mice to characterize potential alterations in the autocrine-paracrine modulation of voltage-dependent calcium channels (VDCCs), which in turn serve to regulate the release of catecholamines. We used mice at the presymptomatic stage (2 months) and mice over 12 months of age, when AD-related cognitive impairment was fully established. We found that the modulation of inward currents through VDCCs induced by extracellular ATP was stronger in CCs isolated from the adrenal medulla of 3xTg mice older than 12 months of age, an effect likely related to disease progression as it was not observed in CCs from age-matched WT mice. This enhanced modulation leads to increased catecholamine release in response to stressful situations, which may explain the non-cognitive behavioral problems found in AD patients.

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 Giβγ 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) → Giβγ → QS → myocyte contractility, offering a universal Vm-GPCR signaling pathway for its functions in the nervous system and other systems containing GPCRs. [ABSTRACT FROM AUTHOR]

Synaptotagmin‐7 (Syt‐7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin‐1 (Syt‐1). Despite a broad appreciation for the importance of Syt‐7, questions remain as to its localization, function in mediating discharge of dense core granule cargos, and role in triggering release in response to physiological stimulation. These questions were addressed using two distinct experimental preparations—mouse chromaffin cells lacking endogenous Syt‐7 (KO cells) and a reconstituted system employing cell‐derived granules expressing either Syt‐7 or Syt‐1. First, using immunofluorescence imaging and subcellular fractionation, it is shown that Syt‐7 is widely distributed in organelles, including dense core granules. Total internal reflection fluorescence (TIRF) imaging demonstrates that the kinetics and probability of granule fusion in Syt‐7 KO cells stimulated by a native secretagogue, acetylcholine, are markedly lower than in WT cells. When fusion is observed, fluorescent cargo proteins are discharged more rapidly when only Syt‐1 is available to facilitate release. To determine the extent to which the aforementioned results are attributable purely to Syt‐7, granules expressing only Syt‐7 or Syt‐1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt‐7 confers substantially greater calcium sensitivity to granule fusion than Syt‐1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium and effects on fusion pore expansion, Syt‐7 plays a central role in regulating secretory output from adrenal chromaffin cells. [ABSTRACT FROM AUTHOR]

Glutamate and its receptors have been demonstrated to promote both basal and nicotine-evoked catecholamine release in bovine chromaffin cells. Multiple glutamate receptors, including metabotropic glutamate receptors (mGluRs), are found in the adrenal glands of several species, as well as in chromaffin cells. However, there is limited information available regarding the expression of glutamate metabotropic receptor (GRM)1-8 mRNAs and the detailed localization of group I mGluRs (mGluR1 and mGluR5) in the rat and human adrenal cortex and medulla. Therefore, we examined mRNA expression of GRM1-8 subunits using reverse transcription-polymerase chain reaction (RT-PCR) and the distribution of mGluR1 and mGluR5 by immunostaining. The results showed that the GRM1-8 mRNAs were expressed in both the cortex and medulla of rat and human adrenal glands with the exception of GRM1 , which was not detectable in the rat adrenal cortex. Immunostaining of mGluR1 revealed that it was localized only in the adrenal medulla of rats but was present in both the adrenal cortex and medulla in humans. In the adrenal medulla, the central part of the adrenal glands, mGluR1 was detected in chromaffin cells but not in nerve fibers and ganglion cells. Immunoactivity of mGluR5 was visible in the capillary wall throughout the adrenal cortex and medulla in rat and human samples. Its immunoactivity was also observed in ganglion cells in the rat adrenal medulla. There was no mGluR5 immunoactivity detected in chromaffin cells and nerve fibers in the rat and human adrenal medulla. Using dissected rat adrenal medulla as a model, we found that treatment with a mGluR1 agonist activated extracellular signal-regulated kinase (ERK) 1/2 and increased the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis. Moreover, these results showed that mGluR1 signaling was involved in hypoxia-induced upregulation of TH in the rat adrenal medulla. This study shows the expression of GRM1-8 mRNAs in rat and human adrenal glands and indicates that glutamate, through the activation of mGluRs, may play various physiological roles in the adrenal gland. Furthermore, mGluR1 may be involved in catecholamine biosynthesis by regulating TH, and mGluR5 may affect cortical and medullar hormone levels by regulating microvascular function. [ABSTRACT FROM AUTHOR]

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 Ca2+‐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. [ABSTRACT FROM AUTHOR]

The ability to detect oxygen availability is a ubiquitous attribute of aerobic organisms. However, the mechanism(s) that transduce oxygen concentration or availability into appropriate physiological responses is less clear and often controversial. This review will make the case for oxygen-dependent metabolism of hydrogen sulfide (H2S) and polysulfides, collectively referred to as reactive sulfur species (RSS) as a physiologically relevant O2 sensing mechanism. This hypothesis is based on observations that H2S and RSS metabolism is inversely correlated with O2 tension, exogenous H2S elicits physiological responses identical to those produced by hypoxia, factors that affect H2S production or catabolism also affect tissue responses to hypoxia, and that RSS efficiently regulate downstream effectors of the hypoxic response in a manner consistent with a decrease in O2. H2S-mediated O2 sensing is then compared to the more generally accepted reactive oxygen species (ROS) mediated O2 sensing mechanism and a number of reasons are offered to resolve some of the confusion between the two.

Lin Chen,1,2 Haitao Xi,1 Juan Xiao,1 Feng Zhang,1 Di Chen,1 Hongyun Huang,1,3 1Cell Therapy Center, Beijing Hongtianji Neuroscience Academy, 2Department of Neurosurgery, Tsinghua University Yuquan Hospital, 3Neurorestoratology Institute, General Hospital of Chinese People’s Armed Police Forces, Beijing, People’s Republic of China Abstract: Neuropathic pain (NP), a common secondary complication following spinal cord injury (SCI), presenting at or below the level of injury is largely refractory to current pharmacological, physical, and surgical treatments. Previous studies have demonstrated the promising value of cell therapy including adrenal chromaffin cells that have the capacity to act as mini-pumps that release amines and peptides for alleviating chronic pain. The paper presents the cases of two gentlemen suffering from severe central NP after thoracic SCI. Six months after chromaffin cell intrathecal injection, their pain relieved significantly. The results demonstrated the preliminary therapeutic efficacy of chromaffin cell transplants in people with NP, and support further research of this treatment strategy for the management of intractable chronic pain due to SCI. Keywords: chromaffin cell, cell transplantation, neuropathic pain, spinal cord injury

Ultraviolet (UV) radiation has a strong biological effect on skin biology, and it switches on adaptive mechanisms to maintain homeostasis in organs such as the skin, adrenal glands, and brain. In this study, we examined the adaptation of the body to repeated bouts of UVB radiation, especially with respect to the catecholamine synthesis pathway of the adrenal glands. The effects of UVB on catecholamine-related enzymes were determined by neurochemical and histological analyses. To evaluate catecholamine changes after chronic excessive UVB irradiation of mouse skin, we examined dopamine and norepinephrine levels in the adrenal glands and blood from UV-irradiated and sham-irradiated mice. We found that chronic excessive UVB exposure significantly reduced dopamine levels in both tissues but did not affect norepinephrine levels. In addition, UVB irradiation significantly increased the levels of related enzymes tyrosine hydroxylase and dopamine-β-hydroxylase. Furthermore, we also found that apoptosis-associated markers were increased and that oxidative defense proteins were decreased, which might have contributed to the marked structural abnormalities in the adrenal medullas of the chronically UVB-irradiated mice. This is the first evidence of the damage to the adrenal gland and subsequent dysregulation of catecholamine metabolism induced by chronic exposure to UVB.

Sphingosine‐1‐phosphate (S1P) is an essential bioactive sphingosine lipid involved in many neurological disorders. Sphingosine kinase 1 (SphK1), a key enzyme for S1P production, is concentrated in presynaptic terminals. However, the role of S1P/SphK1 signaling in exocytosis remains elusive. By detecting catecholamine release from single vesicles in chromaffin cells, we show that a dominant negative SphK1 (SphK1DN) reduces the number of amperometric spikes and increases the duration of foot, which reflects release through a fusion pore, implying critical roles for S1P in regulating the rate of exocytosis and fusion pore expansion. Similar phenotypes were observed in chromaffin cells obtained from SphK1 knockout mice compared to those from wild‐type mice. In addition, extracellular S1P treatment increased the number of amperometric spikes, and this increase, in turn, was inhibited by a selective S1P3 receptor blocker, suggesting extracellular S1P may regulate the rate of exocytosis via activation of S1P3. Furthermore, intracellular S1P application induced a decrease in foot duration of amperometric spikes in control cells, indicating intracellular S1P may regulate fusion pore expansion during exocytosis. Taken together, our study represents the first demonstration that S1P regulates exocytosis through distinct mechanisms: extracellular S1P may modulate the rate of exocytosis via activation of S1P receptors while intracellular S1P may directly control fusion pore expansion during exocytosis. Open science badges: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. [ABSTRACT FROM AUTHOR]

High catecolamine plasma levels because of sympathetic nervous system over‐activity contribute to cirrhosis progression. The aim of this study was to investigate whether chromaffin cells of the adrenal gland might potentiate the deleterious effect exerted by this over‐activity. Electrophysiological patch‐clamp and amperometric experiments with carbon‐fibre electrodes were conducted in single chromaffin cells of control and CCl4‐induced cirrhotic rats. The spontaneous action potential firing frequency was increased in chromaffin cells of cirrhotic rats with respect to control rats. The exocytosis evoked by that firing was also increased. However, exocytosis elicited by ACh did not vary between control and cirrhotic rats. Exocytosis triggered by depolarizing pulses was also unchanged. Amperometric recordings confirmed the lack of increased catecholamine charge released in cirrhosis after ACh or depolarization stimuli. However, the amperometric spikes exhibited faster kinetics of release. The overall Ca2+ entry through voltage‐dependent Ca2+ channels (VDCC), or in particular through Cav1 channels, did not vary between chromaffin cells of control and cirrhotic rats. The inhibition of VDCC by methionine‐enkephaline or ATP was not either altered, but it was increased by adrenaline in cells of cirrhotic rats. When a cocktail composed by the three neurotransmitters was tested in order to approach a situation closer to the physiological condition, the inhibition of VDCC was similar between both types of cells. In summary, chromaffin cells of the adrenal gland might contribute to exacerbate the sympathetic nervous system over‐activity in cirrhosis because of an increased exocytosis elicited by an enhanced spontaneous electrical activity. High catecolamine plasma levels because of sympathetic nervous system over‐activity contribute to cirrhosis progression. The aim of this study was to investigate whether chromaffin cells of the adrenal gland might potentiate this effect. Here we have found that chromaffin cells of the rat adrenal gland might contribute to exacerbate the sympathetic nervous system over‐activity in cirrhosis because of an increased exocytosis elicited by an enhanced spontaneous firing of action potentials. [ABSTRACT FROM AUTHOR]

Recent advances in understanding the intracellular and intercellular features of adrenal chromatin cells as stress transducers are reviewed here, along with their implications for endocrine function in other tissues and organs participating in endocrine regulation in the mammalian organism.

Abstract: 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. [ABSTRACT FROM AUTHOR]

Gasotransmitter hydrogen sulphide (H2S) has emerged as a regulator of multiple physiological and pathophysiological processes throughout. Here, we have investigated the effects of NaHS (fast donor of H2S) and GYY4137 (GYY, slow donor of H2S) on the exocytotic release of catecholamines from fast-perifused bovine adrenal chromaffin cells (BCCs) challenged with sequential intermittent pulses of a K+-depolarizing solution. Both donors caused a concentration-dependent facilitation of secretion. This was not due to an augmentation of Ca2+ entry through voltage-activated Ca2+ channels (VACCs) because, in fact, NaHS and GYY caused a mild inhibition of whole-cell Ca2+ currents. Rather, the facilitation of exocytosis seemed to be associated to an augmented basal [Ca2+]c and the K+-elicited [Ca2+]c transients; such effects of H2S donors are aborted by cyclopiazonic acid (CPA), that causes endoplasmic reticulum (ER) Ca2+ depletion through sarcoendoplasmic reticulum Ca2+ ATPase inhibition and by protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), that impedes the ability of mitochondria to sequester cytosolic Ca2+ during cell depolarization. Inasmuch as CPA and FCCP reversed the facilitation of secretion triggered by K+ in the presence of NaHS and GYY, is seems that such facilitation is tightly coupled to Ca2+ handling by the ER and mitochondria. On the basis of these results, we propose that H2S regulates catecholamine secretory responses triggered by K+ in BCCs by (i) mobilisation of ER Ca2+ and (ii) interference with mitochondrial Ca2+ circulation. In so doing, the clearance of the [Ca2+]c transient will be delayed and the Ca2+-dependent trafficking of secretory vesicles will be enhanced to overfill the secretory machinery with new vesicles to enhance exocytosis. [ABSTRACT FROM AUTHOR]

M-type K+ channels contribute to the resting membrane potential in the sympathetic ganglion neurons of various animals, whereas their expression in adrenal medullary (AM) cells has been controversial. The present experiment aims to explore the expression of M channels comprising the KCNQ2 subunit in the rat AM cell and its immortalized cell line PC12 cells at the protein level and how its expression in PC12 cells is regulated. The KCNQ2 isoform was recognized in homogenates of PC12 cells but not the rat adrenal medullae by immunoblotting and KCNQ2-like immunoreactivity (IR) was detected in PC12 cells but not in rat AM cells. When the PC12 cells were maintained in a dexamethasone-containing medium, KCNQ2-like IR in the cells was suppressed, whereas the removal of fetal bovine serum from the culture medium for 1 day resulted in an increase in KCNQ2-like IR. A similar enhancement occurred when PC12 cells were cultured under conditions where glucocorticoid receptor (GR) and/or mineralocorticoid receptor (MR) activities were suppressed. These morphological findings were confirmed in functional analysis. The cells cultured in the presence of an inhibitor of either GR or MR exhibited larger amplitudes of Ca2+ signal in response to an M channel inhibitor than did the cells in its absence, whereas the resting Ca2+ level in the former was lower than that in the latter. These results indicate that the M channel is not expressed in rat AM cells and this absence of expression may be ascribed to the suppression by glucocorticoid activity. [ABSTRACT FROM AUTHOR]

Pheochromocytomas and paragangliomas (PCC/PGLs) are rare neuroendocrine tumors that are unusually diverse in metabolic profiles, in classes of molecular alterations and across a large number of altered genes. The Cancer Genome Atlas (TCGA) comprehensively profiled the molecular landscape of PCC/PGLs and identified novel genomic alterations and a new molecular classification of PCC/PGLs. In this review, we discuss the significant clinico-molecular findings of this integrated profiling study. We then review the molecular data of the TCGA cohort centering around known markers of sympathoadrenal cell lineage to better understand chromaffin cell biology. This analysis adds a new layer, that of chromaffin cell type, onto the published molecular classifications and in doing so provides inferences about underlying chromaffin cell biology and diversity. [ABSTRACT FROM AUTHOR]

To date, germline or somatic genetic events can be detected for at least 60% of paragangliomas. Strong genotype-phenotype associations have been recognized and become increasingly refined. Characteristics closely linked with genotype include syndromic presentation, age of onset, risk of metastatic disease and predominant anatomic site. In contrast, profiles of catecholamine secretion appear to be largely determined by anatomic location or cell type of origin. This review summarizes current knowledge of genotype-phenotype correlations for paragangliomas in different locations and scrutinizes previous publications on the respective tissues of origin to find potential explanations for site-related differences. We hypothesize that differential sensitivities of distinct chromaffin cell populations to hypoxia are major determinants of these differences, with increased sensitivity to hypoxia likely exacerbating vulnerability to mutation-derived disruption of hypoxic signaling pathways. Potential involvement of endothelin-1, tumor necrosis factor type 1 receptor-associated protein and the hypoxia-inducible miR-210 in the development of abdomino-thoracic or head and neck paragangliomas are discussed. Recognition of factors that predispose to chromosomal losses, or amplify sub-threshold molecular alterations towards tumorigenic events in different (chromaffin) cell types, may facilitate the leap from developing targeted therapies towards establishment of tumor preventative measures. [ABSTRACT FROM AUTHOR]

Chemical signaling strength during intercellular communication can be regulated by secretory cells through controlling the amount of signaling molecules that are released from a secretory vesicle during the exocytosis process. In addition, the chemical signal can also be influenced by the amount of neurotransmitters that is accumulated and stored inside the secretory vesicle compartment. Here, we present the development of analytical methodologies and cell model systems that have been applied in neuroscience research for gaining better insights into the biophysics and the molecular mechanisms, which are involved in the regulatory aspects of the exocytosis machinery affecting the output signal of chemical transmission at neuronal and neuroendocrine cells. [ABSTRACT FROM AUTHOR]

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-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-R activation by endogenous GABA tonically inhibits acetylcholine release from splanchnic nerve terminals and decreases spontaneous Ca fluctuations in CCs, preventing unwanted CA secretion. During intense stress, splanchnic nerve terminals release acetylcholine, which depolarizes CCs and allows the Ca influx that triggers the release of CA and GABA. With time, CA secretion declines, due to voltage-independent inhibition of Cachannels and desensitization of cholinergic nicotinic receptors. Nonetheless, acute activation of GABA-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-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-R-mediated actions are determined by the intracellular chloride concentration ([Cl]) 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. [ABSTRACT FROM AUTHOR]

One of the mechanisms for hypertension is an increase in blood catecholamines due to increased secretion from sympathetic nerve terminals and adrenal medullary chromaffin (AMC) cells. Spontaneously hypertensive rats (SHRs) are used as an animal model of hypertension. Catecholamine secretion in AMC cells occurs in response to humoral factors and neuronal inputs from the sympathetic nerve fibres. Acetylcholine (ACh) released from the nerve terminals activates nicotinic as well as muscarinic ACh receptors. The present experiment aimed to elucidate whether muscarinic receptor–mediated excitation is altered in SHR AMC cells and, if it is, how. Compared with normotensive rat AMC cells, muscarinic stimulation induced greater catecholamine secretion and larger depolarising inward currents in SHR AMC cells. In contrast to normotensive rat AMC cells, the muscarine-induced current consisted of quinine-sensitive and quinine-insensitive components. The former and the latter are possibly ascribed to nonselective cation channel activation and TWIK-related acid-sensitive K+ (TASK) channel inhibition, as noted in guinea pig AMC cells. In fact, immunoreactive material for TASK1 and several isoforms of transient receptor potential canonical (TRPC) channels was detected in SHR AMC cells. Stromal interaction molecule 1 (STIM1), which plays an essential role for heteromeric TRPC1–TRPC4 channel formation and is not expressed in normotensive rat AMC cells, was detected in the cytoplasm and co-localised with TRPC1. The expression of muscarinic M 1 receptors was enhanced in SHR AMC cells compared with normotensive rats. The results indicate that muscarinic excitation is enhanced in SHR AMC cells, probably through facilitation of TRPC channel signalling. [ABSTRACT FROM AUTHOR]

Doc2B is a cytosolic protein with binding sites for Munc13 and Tctex-1 (dynein light chain), and two C2-domains that bind to phospholipids, Ca2+ and SNAREs. Whether Doc2B functions as a calcium sensor akin to synaptotagmins, or in other calcium-independent or calcium-dependent capacities is debated. We here show by mutation and overexpression that Doc2B plays distinct roles in two sequential priming steps in mouse adrenal chromaffin cells. Mutating Ca2+-coordinating aspartates in the C2A-domain localizes Doc2B permanently at the plasma membrane, and renders an upstream priming step Ca2+-independent, whereas a separate function in downstream priming depends on SNARE-binding, Ca2+-binding to the C2B-domain of Doc2B, interaction with ubMunc13-2 and the presence of synaptotagmin-1. Another function of Doc2B – inhibition of release during sustained calcium elevations – depends on an overlapping protein domain (the MID-domain), but is separate from its Ca2+-dependent priming function. We conclude that Doc2B acts as a vesicle priming protein.

Key points The muscarinic acetylcholine receptor (mAChR)-mediated increase in excitability in rat adrenal medullary cells is at least in part due to inhibition of TWIK (tandem of P domains in a weak inwardly rectifying K+ channel)-related acid-sensitive K+ (TASK)1 channels., In this study we focused on the molecular mechanism of mAChR-mediated inhibition of TASK1 channels., Exposure to muscarine resulted in a clathrin-dependent endocytosis of TASK1 channels following activation of the muscarinic M1 receptor (M1R)., This muscarinic signal for the endocytosis was mediated in sequence by phospholipase C (PLC), protein kinase C (PKC), and then the non-receptor tyrosine kinase Src with the consequent tyrosine phosphorylation of TASK1., The present results establish that TASK1 channels are tyrosine phosphorylated and internalized in a clathrin-dependent manner in response to M1R stimulation and this translocation is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells., Abstract Activation of muscarinic receptor (mAChR) in rat adrenal medullary (AM) cells induces depolarization through the inhibition of TWIK-related acid-sensitive K+ (TASK)1 channels. Here, pharmacological and immunological approaches were used to elucidate the molecular mechanism for this mAChR-mediated inhibition. TASK1-like immunoreactive (IR) material was mainly located at the cell periphery in dissociated rat AM cells, and its majority was internalized in response to muscarine. The muscarine-induced inward current and translocation of TASK1 were suppressed by dynasore, a dynamin inhibitor. The muscarinic translocation was suppressed by MT7, a specific M1 antagonist, and the dose-response curves for muscarinic agonist-induced translocation were similar to those for the muscarinic inhibition of TASK1 currents. The muscarine-induced inward current and/or translocation of TASK1 were suppressed by inhibitors for phospholipase C (PLC), protein kinase C (PKC), and/or Src. TASK1 channels in AM cells and PC12 cells were transiently associated with Src and were tyrosine phosphorylated in response to muscarinic stimulation. After internalization, TASK1 channels were quickly dephosphorylated even while they remained in the cytoplasm. The cytoplasmic TASK1-like IR material quickly recycled back to the cell periphery after muscarine stimulation for 0.5 min, but not 10 min. We conclude that M1R stimulation results in internalization of TASK1 channels through the PLC-PKC-Src pathway with the consequent phosphorylation of tyrosine and that this M1R-mediated internalization is at least in part responsible for muscarinic inhibition of TASK1 channels in rat AM cells. [ABSTRACT FROM AUTHOR]

The present study was performed to evaluate the Ca v 1 channel subtypes expressed in human chromaffin cells and the role that these channels play in exocytosis and cell excitability. Here we show that human chromaffin cells obtained from organ donors express Ca v 1.2 and Ca v 1.3 subtypes using molecular and pharmacological techniques. Immunocytochemical data demonstrated the presence of Ca v 1.2 and Ca v 1.3 subtypes, but not Ca v 1.1 or Ca v 1.4. Electrophysiological experiments were conducted to investigate the contribution of Ca v 1 channels to the exocytotic process and cell excitability. Ca v 1 channels contribute to the exocytosis of secretory vesicles, evidenced by the block of 3 μM nifedipine (36.5±2%) of membrane capacitance increment elicited by 200 ms depolarizing pulses. These channels show a minor contribution to the initiation of spontaneous action potential firing, as shown by the 2.5 pA of current at the threshold potential (−34 mV), which elicits 10.4 mV of potential increment. In addition, we found that only 8% of human chromaffin cells exhibit spontaneous action potentials. These data offer novel information regarding human chromaffin cells and the role of human native Ca v 1 channels in exocytosis and cell excitability. [ABSTRACT FROM AUTHOR]

The pro-inflammatory cytokines, tumor necrosis factor-α, and interleukin-1β/α modulate catecholamine secretion, and long-term gene regulation, in chromaffin cells of the adrenal medulla. Since interleukin-6 ( IL6) also plays a key integrative role during inflammation, we have examined its ability to affect both tyrosine hydroxylase activity and adrenomedullary gene transcription in cultured bovine chromaffin cells. IL6 caused acute tyrosine/threonine phosphorylation of extracellular signal-regulated kinase 1/2 ( ERK1/2), and serine/tyrosine phosphorylation of signal transducer and activator of transcription 3 ( STAT3). Consistent with ERK1/2 activation, IL6 rapidly increased tyrosine hydroxylase phosphorylation (serine-31) and activity, as well as up-regulated genes, encoding secreted proteins including galanin, vasoactive intestinal peptide, gastrin-releasing peptide, and parathyroid hormone-like hormone. The effects of IL6 on the entire bovine chromaffin cell transcriptome were compared to those generated by G-protein-coupled receptor ( GPCR) agonists (histamine and pituitary adenylate cyclase-activating polypeptide) and the cytokine receptor agonists (interferon-α and tumor necrosis factor-α). Of 90 genes up-regulated by IL6, only 16 are known targets of IL6 in the immune system. Those remaining likely represent a combination of novel IL6/ STAT3 targets, ERK1/2 targets and, potentially, IL6-dependent genes activated by IL6-induced transcription factors, such as hypoxia-inducible factor 1α. Notably, genes induced by IL6 include both neuroendocrine-specific genes activated by GPCR agonists, and transcripts also activated by the cytokines. These results suggest an integrative role for IL6 in the fine-tuning of the chromaffin cell response to a wide range of physiological and paraphysiological stressors, particularly when immune and endocrine stimuli converge. [ABSTRACT FROM AUTHOR]

It is known that the sustained depolarisation of adrenal medullary bovine chromaffin cells (BCCs) with high K concentrations produces an initial sharp catecholamine release that subsequently fades off in spite depolarisation persists. Here, we have recreated a sustained depolarisation condition of BCCs by treating them with the Na/K ATPase blocker ouabain; in doing so, we searched experimental conditions that permitted the development of a sustained long-term catecholamine release response that could be relevant during prolonged stress. BCCs were perifused with nominal 0Ca solution, and secretion responses were elicited by intermittent application of short 2Ca pulses (Krebs-HEPES containing 2 mM Ca). These pulses elicited a biphasic secretory pattern with an initial 30-min period with secretory responses of increasing amplitude and a second 30-min period with steady-state, non-inactivating responses. The initial phase was not due to gradual depolarisation neither to gradual increases of the cytosolic calcium transients ([Ca]) elicited by 2Ca pulses in BBCs exposed to ouabain; both parameters increased soon after ouabain addition. Νifedipine blocked these responses, and FPL64176 potentiated them, suggesting that they were triggered by Ca entry through non-inactivating L-type calcium channels. This was corroborated by nifedipine-evoked blockade of the L-type Ca channel current and the [Ca] transients elicited by 2Ca pulses. Furthermore, the plasmalemmal Na/Ca exchanger (NCX) blocker SEA0400 caused a mild inhibition followed by a large rebound increase of the steady-state secretory responses. We conclude that these two phases of secretion are mostly contributed by Ca entry through L calcium channels, with a minor contribution of Ca entry through the reverse mode of the NCX. [ABSTRACT FROM AUTHOR]

The circulating catecholamines (CAs) epinephrine (Epi) and norepinephrine (NE) derive from two major sources in the whole organism: the sympathetic nerve endings, which release NE on effector organs, and the chromaffin cells of the adrenal medulla, which are cells that synthesize, store and release Epi (mainly) and NE. All of the Epi in the body and a significant amount of circulating NE derive from the adrenal medulla. The secretion of CAs from adrenal chromaffin cells is regulated in a complex way by a variety of membrane receptors, the vast majority of which are G protein-coupled receptors (GPCRs), including adrenergic receptors (ARs), which act as “presynaptic autoreceptors” in this regard. There is a plethora of CA-secretagogue signals acting on these receptors but some of them, most notably the α 2 ARs, inhibit CA secretion. Over the past few years, however, a few new proteins present in chromaffin cells have been uncovered to participate in CA secretion regulation. Most prominent among these are GRK2 and β-arrestin1, which are known to interact with GPCRs regulating receptor signaling and function. The present review will discuss the molecular and signaling mechanisms by which adrenal chromaffin cell-residing GPCRs and their regulatory proteins modulate CA synthesis and secretion. Particular emphasis will be given to the newly discovered roles of GRK2 and β-arrestins in these processes and particular points of focus for future research will be highlighted, as well. [ABSTRACT FROM AUTHOR]

The neural-crest-derived sympathoadrenal cell lineage gives rise to sympathetic neurons and to endocrine chromaffin cells of the adrenal medulla. Both cell types express a largely overlapping set of genes, including those coding for the molecular machinery related to the synthesis and exocytotic release of catecholamines. During their early development, sympathetic neurons and chromaffin cells rely on a shared transcription factor network that controls the establishment of these common features. Despite many similarities, mature sympathetic neurons and chromaffin cells significantly differ regarding their morphology and function. Most prominently, sympathetic neurons possess axons that are absent in mammalian adrenal chromaffin cells. The molecular mechanism underlying the divergent development of sympathoadrenal cells into neuronal and endocrine cells remains elusive. Mutational inactivation of the ribonuclease dicer hints at the importance of microRNAs in this diversification. We show here that miR-124 is detectable in developing sympathetic neurons but absent in chromaffin cell precursors. We further demonstrate that miR-124 promotes neurite elongation when transfected into cultured chromaffin cells indicating its capability to support the establishment of a neuronal morphology in non-neuronal sympathoadrenal cells. Our results also show that treatment of PC12 cells with the neurotrophin nerve growth factor leads to an upregulation of miR-124 expression and that inhibition of miR-124 reduces nerve-growth-factor-induced neurite outgrowth in PC12 cells. Thus, our data indicate that miR-124 contributes to the establishment of specific neuronal features in developing sympathoadrenal cells. [ABSTRACT FROM AUTHOR]

Using fluorescence [Ca] imaging in rat adrenal slices, we characterized the effects of agonists and antagonists of the GABA receptor (GABA-R) on resting intracellular Ca ([Ca]) and spontaneous [Ca] fluctuations (SCFs) in hundreds of individual chromaffin cells (CCs) recorded simultaneously in situ. Muscimol, a GABA-R agonist (20 μM; 25 s), induced an increase of resting [Ca] in 43 ± 3 % of CCs, a decrease in 26 ± 2 %, and no response in 30 ± 5 %. In Ca-free external medium, SCFs ceased completely and muscimol failed to elicit [Ca] rises. All muscimol-induced [Ca] changes were blocked by the GABA-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 GABA-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 GABA-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. [ABSTRACT FROM AUTHOR]

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 Ca2+ mobilization from intracellular stores; rather, IG20-elicited secretion entirely dependent on Ca2+ entry through L-subtype voltage-activated Ca2+ 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, Ca2+ 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. [ABSTRACT FROM AUTHOR]

Adrenal chromaffin cells (CCs) from spontaneously hypertensive rats (SHRs) secrete more catecholamine (CA) upon stimulation than CCs from normotensive Wistar Kyoto rats (WKY). Unitary CA exocytosis events, both spontaneous and stimulated, were amperometrically recorded from cultured WKY and SHR CCs. Both strains display spontaneous amperometric spikes but SHR CCs produce more spikes and of higher mean amplitude. After a brief stimulation with high K or caffeine which produces voltage-gated Ca influx or intracellular Ca release, respectively, more spikes and of greater mean amplitude and unitary charge were recorded in SHR CCs. Consequently, peak cumulative charge was ~2-fold higher in SHR CCs. Ryanodine (10 μM), a specific blocker of the ryanodine receptors reduced depolarization-induced peak cumulative charge by ~10 % in WKY and ~77 % in SHR CCs, suggesting, a larger contribution of Ca-induced Ca release to CA exocytosis in SHR CCs. Accordingly, Ca imaging showed larger [Ca] signals induced both by depolarization and caffeine in SHR CCs. Distribution amplitude histograms showed that small amperometric spikes (0-50 pA) are more frequent in WKY than in SHR CCs. Conversely, medium (50-190 pA) and large (190-290 pA) spikes are more numerous in SHR than in WKY CCs. This study reveals that the enhanced CA secretion in SHR CCs results from a combination of (1) larger depolarization-induced Ca transients, due to a greater Ca-induced intracellular Ca release, (2) more exocytosis events per time unit, and (3) a greater proportion of medium and large amperometric spikes probably due to a higher mean CA content per granule. Enhanced CA release by excessive amplification by Ca induced Ca release and larger granule catecholamine content contributes to the increased CA plasma levels and vasomotor tone in SHRs. [ABSTRACT FROM AUTHOR]