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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]

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]

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]

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]

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]

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]

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 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]

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]

We have previously shown that Regulator of Calcineurin 1 ( RCAN1) regulates multiple stages of vesicle exocytosis. However, the mechanisms by which RCAN1 affects secretory vesicle exocytosis and quantal release kinetics remain unknown. Here, we use carbon fibre amperometry to detect exocytosis from chromaffin cells and identify these underlying mechanisms. We observe reduced exocytosis with repeated stimulations in chromaffin cells over-expressing RCAN1 ( RCAN1ox), but not in wild-type ( WT) cells, indicating a negative effect of RCAN1 on vesicle recycling and endocytosis. Acute exposure to calcineurin inhibitors, cyclosporine A and FK-506, replicates this effect in WT cells but has no additional effect in RCAN1ox cells. When we chronically expose WT cells to cyclosporine A and FK-506 we find that catecholamine release per vesicle and pre-spike foot ( PSF) signal parameters are decreased, similar to that in RCAN1ox cells. Inhibiting calcineurin activity in RCAN1ox cells has no additional effect on the amount of catecholamine release per vesicle but further reduces PSF signal parameters. Although electron microscopy studies indicate these changes are not because of altered vesicle number or distribution in RCAN1ox cells, the smaller vesicle and dense core size we observe in RCAN1ox cells may underlie the reduced quantal release in these cells. Thus, our results indicate that RCAN1 most likely affects vesicle recycling and quantal release kinetics via the inhibition of calcineurin activity. [ABSTRACT FROM AUTHOR]

The four Rab3 paralogs A-D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here, we used a quadruple Rab3 knockout (KO) ( rab3a, rab3b, rab3c, rab3d null, here denoted as ABCD−/−) mouse line to investigate Rab3 function in embryonic mouse adrenal chromaffin cells by electron microscopy and electrophysiological measurements. We show that in cells from ABCD−/− animals large dense-core vesicles (LDCVs) are less abundant, while the number of morphologically docked granules is normal. By capacitance measurements, we show that deletion of Rab3s reduces the size of the releasable vesicle pools but does not alter their fusion kinetics, consistent with an altered function in vesicle priming. The sustained release component has a sigmoid shape in ABCD−/− cells when normalized to the releasable pool size, indicating that vesicle priming follows at a higher rate after an initial delay. Rescue experiments showed that short-term (4-6 h) overexpression of Rab3A or Rab3C suffices to rescue vesicle priming and secretion, but it does not restore the number of secretory vesicles. We conclude that Rab3 proteins play two distinct stimulating roles for LDCV fusion in embryonic chromaffin cells, by facilitating vesicle biogenesis and stabilizing the primed vesicle state. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 114, 617–626. Neurons and certain kinds of endocrine cells, such as adrenal medullary (AM) cells, have large dense-core vesicles (LDCVs) and synaptic vesicles or synaptic-like microvesicles (SLMVs). These secretory vesicles differ in Ca2+ sensitivity and contain different signaling substances. We have recently reported that GABA functions as a paracrine factor in rat AM cells and modulates catecholamine secretion. The present experiment was undertaken to examine the subcellular localization of the GABA system including GABA itself in AM cells. Fractionation analysis with sucrose density gradient and immunocytochemistry indicated that vesicular GABA transporter (VGAT) was localized in LDCVs and not SLMVs in rat and bovine AM cells. In addition, significant amounts of GABA were detected in high density fractions, which contained LDCVs. When green fluorescence protein-VGAT and green fluorescence protein-vesicular ACh transporter were exogenously expressed in PC12 cells, the former and the latter were selectively targeted to LDCVs and SLMVs, respectively. We conclude that GABA is stored in chromaffin granules in rat and bovine AM cells through VGAT. [ABSTRACT FROM AUTHOR]

Of all the intracellular organelles, secretory granules contain by far the highest calcium concentration; secretory granules of typical neuroendocrine chromaffin cells contain ∼40 mM Ca2+ and occupy ∼20% cell volume, accounting for >60% of total cellular calcium. They also contain the majority of cellular inositol 1,4,5-trisphosphate receptors (IP3Rs) in addition to the presence of >2 mM of chromogranins A and B that function as high-capacity, low-affinity Ca2+ storage proteins. Chromogranins A and B also interact with the IP3Rs and activate the IP3R/Ca2+ channels. In experiments with both neuroendocrine PC12 and nonneuroendocrine NIH3T3 cells, in which the number of secretory granules present was changed by either suppression or induction of secretory granule formation, secretory granules were demonstrated to account for >70% of the IP3-induced Ca2+ releases in the cytoplasm. Moreover, the IP3 sensitivity of secretory granule IP3R/Ca2+ channels is at least ∼6- to 7-fold more sensitive than those of the endoplasmic reticulum, thus enabling secretory granules to release Ca2+ ahead of the endoplasmic reticulum. Further, there is a direct correlation between the number of secretory granules and the IP3 sensitivity of cytoplasmic IP3R/Ca2+ channels and the increased ratio of IP3-induced cytoplasmic Ca2+ release, highlighting the importance of secretory granules in the IP3-dependent Ca2+ signaling. Given that secretory granules are present in all secretory cells, these results presage critical roles of secretory granules in the control of cytoplasmic Ca2+ concentrations in other secretory cells [ABSTRACT FROM AUTHOR]

Large conductance calcium-activated potassium (BK) channels are very prominently expressed in adrenal chromaffin and many anterior pituitary cells, where they shape intrinsic excitability complexly. Stress- and sex-steroids regulate alternative splicing of Slo-α, the pore-forming subunit of BK channels, and chronic behavioural stress has been shown to alter Slo splicing in tree shrew adrenals. In the present study, we focus on mice, measuring the effects of chronic behavioural stress on total mRNA expression of the Slo-α gene, two key BK channel β subunit genes (β2 and β4), and the ‘STREX’ splice variant of Slo-α. As a chronic stressor, males of the relatively aggressive SJL strain were housed with a different unfamiliar SJL male every 24 h for 19 days. This ‘social-instability’ paradigm stressed all individuals, as demonstrated by reduced weight gain and elevated corticosterone levels. Five quantitative reverse transcriptase-polymerase chain assays were performed in parallel, including β-actin, each calibrated against a dilution series of its corresponding cDNA template. Stress-related changes in BK expression were larger in mice tested at 6 weeks than 9 weeks. In younger animals, Slo-α mRNA levels were elevated 44% and 116% in the adrenal medulla and pituitary, respectively, compared to individually-housed controls. β2 and β4 mRNAs were elevated 162% and 194% in the pituitary, but slightly reduced in the adrenals of stressed animals. In the pituitary, dominance scores of stressed animals correlated negatively with α and β subunit expression, with more subordinate individuals exhibiting levels that were three- to four-fold higher than controls or dominant individuals. STREX variant representation was lower in the subordinate subset. Thus, the combination of subunits responding to stress differs markedly between adrenal and pituitary glands. These data suggest that early stress will differentially affect neuroendocrine cell excitability, and call for detailed analysis of functional consequences. [ABSTRACT FROM AUTHOR]

The muscarinic receptor is known to be involved in the acetylcholine-induced secretion of catecholamines in the adrenal medulla (AM) cells of various mammals. The ionic mechanisms, however, have not been elucidated yet. Thus, we investigated the issue in acutely isolated rat AM cells with the perforated patch clamp method. Bath application of 30 μM muscarine induced depolarization with the consequent generation of action potentials or an inward current at negative membrane potentials. The muscarine-sensitive current instantaneously changed in amplitude upon application of command pulses without a time-dependent component, altered the polarity as a K+-electrode, and showed rectification of the Goldman-Hodgkin-Katz (GHK) type. The whole-cell current at −20 mV was inhibited by external H+ ions with a concentration responsible for half inhibition of pH 7.09 and muscarine failed to induce a further inward current during exposure to a saline in which pH decreased to 6.5. A similar occlusion occurred in secretion when pH in muscarine-containing saline decreased to 6.6. RT-PCR, immunoblotting, and immunocytochemistry suggested that rat AM cells mainly express the TASK1 channel. This TASK channel in AM cells may directly sense a decrease in blood pH, which occurs during exercise. The muscarine action was mimicked by oxotremorine–methiodide, but not by oxotremorine. The present results indicate that activation of muscarinic receptors or a decrease in external pH in the rat AM cell induces secretion through the inhibition of TASK1-like channels. [ABSTRACT FROM AUTHOR]

Protein kinase C (PKC) activity mediates multiple neurosecretory processes, but these are poorly understood due in part to the existence of at least 12 PKC isoforms. Using amperometry to record quantal catecholamine release from chromaffin cells, we found that both broad spectrum PKC antagonists and rottlerin, a selective inhibitor of the novel isoforms PKC θ and PKC δ, decreased quantal size and the number of secretory events recorded per stimulus. In contrast, drugs that selectively inhibit the atypical and conventional PKC isoforms had no effect on these parameters. While both PKC θ and δ were expressed in chromaffin cells, mice deficient for PKC θ, but not for PKC δ, exhibited lower quantal size than wild-type and were insensitive to rottlerin. Finally, an inhibitory PKC θ pseudosubstrate produced rottlerin-like responses in wild-type mice, indicating that the lack of rottlerin response in the PKC θ mutants was not the result of a form of compensation. These findings demonstrate neurosecretory regulation by a novel PKC isoform, PKC θ, and should contribute to defining mechanisms of activity-dependent regulation of neurosecretion. [ABSTRACT FROM AUTHOR]

Various studies have focused in the relative contribution of different voltage-activated Ca2+ channels (VACC) to total transmitter release. However, how Ca2+ entry through a given VACC subtype defines the pattern of individual exocytotic events remains unknown. To address this question, we have used amperometry in bovine chromaffin cells. L, N, and P/Q channels were individually or jointly blocked with furnidipine, ω-conotoxin GVIA, ω-agatoxin IVA, or ω-conotoxin MVIIC. The three channel types contributed similarly to cytosolic Ca2+ signals induced by 70 mmol/L K+. However, they exhibited different contributions to the frequency of exocytotic events and they were shown to differently regulate the final steps of the exocytosis. When compared with the other VACC subtypes, Ca2+ entry through P/Q channels effectively induced exocytosis, it decreased fusion pore stability and accelerated its expansion. Conversely, Ca2+ entry through N channels was less efficient in inducing exocytotic events, also slowing fusion pore expansion. Finally, Ca2+ entry through L channels inefficiently induced exocytosis, and the individual blockade of this channel significantly modified fusion pore dynamics. The distance between a given VACC subtype and the release sites could account for the differential effects of the distinct VACC on the fusion pore dynamics. [ABSTRACT FROM AUTHOR]

Bioactive lysophospholipids (LPLs) are released by blood cells and can modulate many cellular activities such as angiogenesis and cell survival. In this study, the effects of sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) on excitability and exocytosis in bovine chromaffin cells were investigated using the whole-cell configuration of the patch-clamp. Voltage-gated Ca2+ current was inhibited by S1P and LPA pre-treatment in a concentration-dependent manner with IC50s of 0.46 and 0.79 μmol/L, respectively. Inhibition was mostly reversible upon washout and prevented by suramin, an inhibitor of G-protein signaling. Na+ current was inhibited by S1P, but not by LPA. However, recovery of Na+ channels from inactivation was slowed by both LPLs. The outward K+ current was also significantly reduced by both LPLs. Chromaffin cells fired repetitive action potentials in response to minimal injections of depolarizing current. Repetitive activity was dramatically reduced by LPLs. Consistent with the reduction in Ca2+ current, exocytosis elicited by a train of depolarizations and the ensuing endocytosis were both inhibited by LPL pre-treatments. These data demonstrate the interaction between immune and endocrine systems mediated by the inhibitory effects of LPLs on the excitability of adrenal chromaffin cells. [ABSTRACT FROM AUTHOR]

Decrease in connexin (Cx) expression and loss of gap junctional intercellular communication (GJIC) have been associated with aberrant cell growth and enhanced neoplastic phenotype. We studied the expression of Cx26, Cx32, Cx43, and Cx50 in chromaffin cells of 10 normal human adrenal glands, 10 benign, and 13 malignant pheochromocytomas. Immunohistochemistry showed that Cx50 expression seemed to be the predominant form of Cx expressed in human chromaffin cells, whereas Cx43 immunoreactivity was the most prominent form found in the adrenal cortex. However, Western blotting revealed that nonphosphorylated and singly and doubly phosphorylated forms of Cx43 were present within the malignant adrenomedullary cells at the same time. Cx26 and Cx32 were distributed inhomogeneously with no emphasis of expression in the types of tissues studied. Cx50 expression was diminished in malignant pheochromocytomas. We conclude that there is a differential expression of Cx in human adrenals. Immunohistological testing for Cx expression does not however allow differentiation of benign from malignant pheochromocytomas. [ABSTRACT FROM AUTHOR]

Individual exocytic events in intact adrenal medulla were visualized by two-photon extracellular polar-tracer imaging. Exocytosis of chromaffin vesicles often occurred in a sequential manner, involving first vesicles located at the cell periphery and then those present deeper within the cytoplasm. Sequential exocytosis occurred preferentially at regions of the plasma membrane facing the intercellular space. The compound vesicles swelled to more than five times their original volume and formed vacuolar exocytic lumens as a result of expansion of intravesicular gels and their confinement within the lumen by the fusion pore and the narrow intercellular space. Such luminal swelling greatly promoted sequential exocytosis. The SNARE protein SNAP25 rapidly migrated from the plasma membrane to the membrane of fused vesicles. These data indicate that vesicles present deeper within the cytoplasm can be fusion ready like those at the cell periphery, and that swelling of exocytic lumens promotes assembly of the fusion machinery. We suggest the existence of two molecular configurations for fusion-ready states in Ca2+-dependent exocytosis. [ABSTRACT FROM AUTHOR]

Abstract: The major secretory granule proteins chromogranins A (CGA) and B (CGB) have recently been shown to play critical roles in inositol 1,4,5-trisphosphate-dependent intracellular Ca2+ mobilizations. We determined here the subcellular distribution of CGA and CGB based on 3D-images of chromaffin cells, and found that ∼95% of cellular CGA was present in secretory granules while ∼5% was in the endoplasmic reticulum (ER), whereas ∼57% of cellular CGB was in secretory granules while ∼24% and ∼19% were in the ER and nucleus, respectively. These results suggest that chromogranins are at the center of intracellular Ca2+ homeostasis in secretory cells. [Copyright &y& Elsevier]

Abstract: Given the importance of inositol 1,4,5-trisphosphate receptor (IP3R)/Ca2+ channels in the control of intracellular Ca2+ concentrations, we determined the relative concentrations of the IP3R isoforms in subcellular organelles, based on serially sectioned electron micrographs. The endoplasmic reticulum (ER) was estimated to contain 15–20% of each of the three IP3R isoforms while secretory granules contained 58–69%. The nucleus contained ∼15% each of IP3R-1 and -2, but 25% of IP3R-3, whereas the plasma membrane contained ∼1% or less of each. These suggested that secretory granules, the nucleus and ER are at the center of IP3-dependent intracellular Ca2+ control mechanisms in chromaffin cells. [Copyright &y& Elsevier]

We have previously shown that the phosphorylation of Ser19 in tyrosine hydroxylase can increase the rate of phosphorylation of Ser40 in tyrosine hydroxylase threefold in vitro. In this report we investigated the role of Ser19 on Ser40 phosphorylation in intact cells. Treatment of bovine chromaffin cells with anisomycin produced a twofold increase in Ser19 phosphorylation with no increase in Ser31 phosphorylation and only a small increase in Ser40 phosphorylation. Treatment of bovine chromaffin cells with forskolin produced a fourfold increase in Ser40 phosphorylation but no significant increase in either Ser19 or Ser31 phosphorylation. When chromaffin cells were first treated with anisomycin, the level of Ser40 phosphorylation after treatment by forskolin was 76% greater than the level of Ser40 phosphorylation in cells treated with forskolin alone. This potentiation of Ser40 phosphorylation by anisomycin could be completely blocked by the p38 MAP (mitogen-activated protein) kinase inhibitor SB 203580. The potentiation of Ser40 phosphorylation by anisomycin was not due to an increase in Ser40 kinase activity. Anisomycin treatment of chromaffin cells potentiated the forskolin-induced increase in tyrosine hydroxylase activity by 50%. This potentiation of activity was also blocked by SB 203580. These data provide the first evidence that the phosphorylation of Ser19 can potentiate the phosphorylation of Ser40 and subsequent activation of tyrosine hydroxylase in intact cells. [ABSTRACT FROM AUTHOR]

Abstract The presence of myosin II and V in chromaffin cells and their subcellular distribution is described. Myosin II and V distribution in sucrose density gradients showed only a strong correlation between the distribution of myosin V and secretory vesicle markers. Confocal microscopy images demonstrated colocalization of myosin V with dopamine β-hydroxylase, a chromaffin vesicle marker, whereas myosin II was present mainly in the cell cortex. Cell depolarization induced, in a Ca[sup 2+] and time-dependent manner, the dissociation of myosin V from chromaffin vesicles suggesting that this association was not permanent but determined by secretory cycle requirements. Myosin II was also found in the crude granule fraction, however, its distribution was not affected by cell depolarization. Myosin V head antibodies were able to inhibit secretion whereas myosin II antibodies had no inhibitory effect. The pattern of inhibition indicated that these treatments interfered with the transport of vesicles from the reserve to the release-ready compartment, suggesting the involvement of myosin V and not myosin II in this transport process. The results described here suggest that myosin V is a molecular motor involved in chromaffin vesicle secretion. However, these results do not discard an indirect role for myosin II in secretion through its interaction with F-actin networks. [ABSTRACT FROM AUTHOR]

Phospholipase D (PLD) has been proposed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. We recently described the activation of an ADP ribosylation factor-regulated PLD at the plasma membrane of chromaffin cells undergoing secretagogue-stimulated exocytosis. We show here that the isoform involved is PLD1b, and, using a real-time assay for individual cells, that PLD activation and exocytosis are closely correlated. Moreover, overexpressed PLD1, but not PLD2, increases stimulated exocytosis in a phosphatidyl-inositol 4,5-bisphosphate-dependent manner, whereas catalytically inactive PLD1 inhibits it. These results provide the first direct evidence that PLD1 is an important component of the exocytotic machinery in neuroendocrine cells. [ABSTRACT FROM AUTHOR]

Abstract: Bovine chromaffin cells were microencapsulated within alginate-polylysine-alginate (APA) membranes. Microencapsulated bovine chromaffin cells as well as unencapsulated cells and empty microcapsules were grafted into the brain of hemiparkinsonian rats with 6-hydroxydopamine (6-OHDA) lesions. Apomorphine-induced rotational behavior of the host animals and the survival of the grafted chromaffin cells were examined after transplantation. The animals receiving microencapsulated bovine chromaffin cells showed a significant decrease (17.6–35.6%) in apomorphine-induced rotation 1 week postimplantation that remained stable for the 10 month test period. Fluorescent histochemistry further revealed that microencapsulation increased the chromaffin cell survival with only a minimum host reaction for up to 10 months posttransplantation while the survival of free, unencapsulated chromaffin cells was only modest and was accompanied by a large inflammatory response. The reduction of apomorphine-induced rotations was correlated with the survival of bovine chromaffin cells in the host brain. The data indicate that encapsulation of bovine chromaffin cells in APA membranes reduces the host immune response to the xenograft and prolongs the viability of the grafted cells. [ABSTRACT FROM AUTHOR]

AbstractThe mammalian adrenal gland consists of two anatomically distinct parts: an outer cortex that synthesizes steroids and a central medulla that contains catecholamine-producing chromaffin cells. Although derived from different embryological origins, the two secretory tissues in the adult animal are functionally as well as structurally linked. Glucocorticoids, a class of steroid hormones produced by the cortex, exert a variety of effects on medullary chromaffin cells. They modulate the expression of specific genes via activation of glucocorticoid receptors that act as transcription factors and either up- or down-regulate mRNA synthesis. The direct binding to and modulation of cation channels by glucocorticoids as well as the control of mRNA or protein stability are other proposed mechanisms of glucocorticoid action. The activity of phenylethanolamine N-methyltransferase, the enzyme that converts noradrenaline into adrenaline, is stimulated by glucocorticoids, which causes the conversion of noradrenergic to adrenergic chromaffin cells. Other phenotypic manifestations of glucocorticoid action include the upregulation of catecholamine synthesis, storage, and secretion. Furthermore, glucocorticoids have been implicated in chromaffin cell differentiation. However, recent gene knockout experiments suggest that glucocorticoid signalling is required only for the acquisition of the adrenergic but not the noradrenergic phenotype. [ABSTRACT FROM AUTHOR]

Chromaffin cell secretion requires cortical F-actin disassembly and it has been suggested that scinderin, a Ca[sup 2+]-dependent F-actin severing protein, controls cortical actin dynamics. An antisense oligodeoxynucleotide targeting the scinderin gene was used to decrease the expression of the protein and access its role in secretion. Treatment with 2 µm scinderin antisense oligodeoxynucleotide for 4 days produced a significant decrease in scinderin expression and its mRNA levels. The expression of gelsolin, another F-actin severing protein, was not affected. Scinderin decrease was accompanied by concomitant and parallel decreases in depolarization-evoked cortical F-actin disassembly and exocytosis. Similar treatment with a mismatched oligodeoxynucleotide produced no effects. Scinderin antisense oligodeoxynucleotide treatment was also a very effective inhibitor of exocytosis in digitonin-permeabilized cells stimulated with increasing concentrations of Ca[sup 2+]. This ruled out scinderin antisense interference with stimulation-induced depolarization or Ca[sup 2+] channel activation. Scinderin antisense treatment decreased the maximum (B[sub max]) secretory response to Ca[sup 2+] without modifying the affinity (K[sub m]) of the cation for the exocytotic machinery. Moreover, the antisense treatment did not affect norepinephrine uptake or the expression of dopamine β-hydroxylase, suggesting that the number and function of chromaffin vesicles was not modified. In addition, scinderin antisense treatment did not alter the expression of proteins involved in vesicle–plasma membrane fusion, such as synaptophysin, synaptotagmin or syntaxin, indicating a lack of effects on the fusion machinery components. These observations strongly suggest that scinderin is a key player in the events involved in the secretory process. [ABSTRACT FROM AUTHOR]

Abstract: In this study, the relationship between intracellular calcium stores and depolarization-evoked stimulation was examined in bovine chromaffin cells, using changes in membrane capacitance to monitor both exocytosis and endocytosis. Cells were voltage-clamped using the perforated whole-cell patch configuration to minimize alterations in intracellular constituents. Control cells exhibited reproducible secretory responses each time the cell was stimulated. However, the same stimulation protocol elicited progressively smaller secretory responses in cells where their intracellular calcium store was emptied by thapsigargin. Transient elevation of the intracellular calcium concentration with a brief histamine treatment enhanced subsequent secretory responses in control but not in thapsigargin-treated cells. A series of depolarizations to -20 mV, which allowed small amounts of Ca2+ influx but which by itself did not trigger catecholamine secretion, enhanced subsequent exocytosis in both control and thapsigargin-treated cells. Caffeine-pretreated cells exhibited a rundown in the secretory response that was similar to that produced by thapsigargin. These results suggest that brief elevations of [Ca2+]i could enhance subsequent secretory responses. In addition, the data suggest that intracellular calcium stores are vital for the maintenance of exocytosis during repetitive stimulation. [ABSTRACT FROM AUTHOR]

SUMMARY1. The goal of the present review is to recount the evidence that endothelin (ET) has a significant influence on the peripheral sympathetic nervous system by regulating the function of the adrenal medulla. 2. The presence of an active ET system in the adrenal medulla has been clearly demonstrated. Endothelin protein, mRNA, binding sites and ET-converting enzyme have been identified in adrenal tissue and medullary chromaffin cells, suggesting that this peptide may contribute to the regulation of adrenal medullary function. 3. Studies investigating the function of ET in the adrenal gland have demonstrated that ET has a stimulatory effect on the adrenal medulla. Endothelin elicits an increase in catecholamine release from perfused intact adrenal glands as well as from cultured chromaffin cells. This effect has been shown to be mediated by ETA and ETB receptors. 4. The mechanism by which ET causes an increase in catecholamine release from the adrenal medulla appears to be independent of cholinergic activation of chromaffin cells. Endothelin has been shown to act directly at chromaffin cells to increase intracellular calcium, which results in catecholamine release. 5. Endothelin can indirectly affect catecholamine release by its effect on adrenal blood flow. Studies indicate that ET has both vasoconstrictor and vasodilator effects in the adrenal gland, which suggests a role for ET in the regulation of adrenal blood flow. Endothelin has also been proposed to participate in the selective contraction of the adrenomedullary veins, which enhances the discharge of catecholamines from the adrenal gland during activation. [ABSTRACT FROM AUTHOR]

Sodium arsenite and osmotic shock both stimulated stress-activated protein kinase-2 (SAPK2, also termed RK, p38, CSBP and Mxi2) and its downstream target mitogen-activated protein kinase (MAP kinase)-activated protein kinase-2 (MAPKAP-K2) in bovine adrenal chromaffin and rat PC12 cells. The same stimuli also increased tyrosine hydroxylase activity 2-3-fold and induced its phosphorylation at Ser19, a residue phosphorylated by MAPKAP-K2 in vitro. The arsenite-induced activation of tyrosine hydroxylase and its phosphorylation at Ser19 were prevented by SB 203580 at concentrations similar to those that inhibited SAPK2 in vitro. These results indicate that MAPKAP-K2 mediates the stress-induced activation of tyrosine hydroxylase. SB 203580 had no effect on the phosphorylation or activation of tyrosine hydroxylase induced by nerve growth factor or forskolin, which trigger the phosphorylation of Ser31 and Ser40, respectively. Stimulation of bovine adrenal chromaffin cells with acetylcholine activated SAPK2 and MAPKAP-K2, as well as p42/p44 MAP kinases and their downstream target MAPKAP-K1. The half-times for activation of MAPKAP-K1 and MAPKAP-K2 (1 min) were similar. In contrast, the activation of tyrosine hydroxylase by acetytcholine peaked within t rain and gradually declined thereafter. Neither SB 203580 (which blocked the activation of MAPKAP-K2 by acetylcholine) nor PD 98059 (which prevented the activation of p42/p44 MAP kinases by acetylcholine) affected tyrosine hydroxylase activation after 1 min, but these compounds inhibited activation by 40-50% after 5 min. PD 98059 prevented the acetylcholine-induced phosphorylation of tyrosine hydroxylase at Ser31, the residue targetted by p42/p44 MAP kinases in vitro, but did not inhibit the phosphorylation of Ser40 (which is phosphorylated by MAPKAP-K1 in vitro). Our results establish that p42/p44 MAP kinases mediate the acetylcholine-induced phosphorylation of tyrosine hydroxylase at Set31. SB 203580 did not suppress the phosphorylation of Ser19 or Ser40 by acetylcholine but, like PD 98059, this drug decreased the phosphorylation of Ser31. SAPK2 may therefore contribute to the acetylcholine-induced activation of tyrosine hydroxylase by facilitating (in an unknown way) its phosphorylation by MAP kinases. [ABSTRACT FROM AUTHOR]

AbstractThe effects of activation of muscarinic receptors on chromaffin cells and splanchnic nerve terminals were studied in a rat adrenal slice preparation. In chromaffin cells, muscarine induced a transient hyperpolarization followed by a depolarization associated with cell spiking. The hyperpolarization was blocked by charybdotoxin (1 μm) and tetraethylammonium chloride (TEA, 1 mm), but was not affected by 200 μm Cd2+ or removal of external Ca2+, consistent with activation of BK channels. This would follow internal Ca2+ mobilization, as shown by Ca2+ imaging with fura‐2 on isolated chromaffin cells in culture. Under voltage‐clamp, outward BK currents were insensitive to MT3 toxin, a specific muscarinic m4 receptor antagonist. In contrast, muscarine‐induced depolarization was due to a m4 receptor‐mediated inward current blocked by MT3 toxin. This current was permeable to cations and was associated with Ca2+ entry and subsequently, Ca2+‐induced Ca2+ release. Finally, both muscarine (25 μm) and oxotremorine (10 μm) decreased the amplitude and frequency of KCl‐evoked excitatory postsynaptic currents, without affecting quantal size, consistent with a presynaptic inhibitory effect. Taken together, our data suggest that activation of m4 and probably m3 muscarinic receptors results in a strong, long‐lasting excitation of chromaffin cells, as well as an uncoupling of synaptic inputs onto these cells. [ABSTRACT FROM AUTHOR]