Nicotinic Receptors in Human Chromaffin Cells: Characterization, Functional and Physical Interactions between Subtypes and Regulation.

This review summarizes our research on nicotinic acetylcholine receptors in human chromaffin cells. Limited research has been conducted in this field on human tissue, primarily due to the difficulties associated with obtaining human cells. Receptor subtypes were characterized here using molecular biology and electrophysiological patch-clamp techniques. However, the most significant aspect of this study refers to the cross-talk between the two main subtypes identified in these cells, the α7- and α3ß4* subtypes, aiming to avoid their desensitization. The article also reviews other aspects, including the regulation of their expression, function or physical interaction by choline, Ca2+, and tyrosine and serine/threonine phosphatases. Additionally, the influence of sex on their expression is also discussed.

Adaptive remodeling of the stimulus-secretion coupling: Lessons from the 'stressed' adrenal medulla.

Stress is part of our daily lives and good health in the modern world is offset by unhealthy lifestyle factors, including the deleterious consequences of stress and associated pathologies. Repeated and/or prolonged stress may disrupt the body homeostasis and thus threatens our lives. Adaptive processes that allow the organism to adapt to new environmental conditions and maintain its homeostasis are therefore crucial. The adrenal glands are major endocrine/neuroendocrine organs involved in the adaptive response of the body facing stressful situations. Upon stress episodes and in response to activation of the sympathetic nervous system, the first adrenal cells to be activated are the neuroendocrine chromaffin cells located in the medullary tissue of the adrenal gland. By releasing catecholamines (mainly epinephrine and to a lesser extent norepinephrine), adrenal chromaffin cells actively contribute to the development of adaptive mechanisms, in particular targeting the cardiovascular system and leading to appropriate adjustments of blood pressure and heart rate, as well as energy metabolism. Specifically, this chapter covers the current knowledge as to how the adrenal medullary tissue remodels in response to stress episodes, with special attention paid to chromaffin cell stimulus-secretion coupling. Adrenal stimulus-secretion coupling encompasses various elements taking place at both the molecular/cellular and tissular levels. Here, I focus on stress-driven changes in catecholamine biosynthesis, chromaffin cell excitability, synaptic neurotransmission and gap junctional communication. These signaling pathways undergo a collective and finely-tuned remodeling, contributing to appropriate catecholamine secretion and maintenance of body homeostasis in response to stress.

All SNAP25 molecules in the vesicle-plasma membrane contact zone change conformation during vesicle priming.

In neuronal cell types, vesicular exocytosis is governed by the SNARE (soluble NSF attachment receptor) complex consisting of synaptobrevin2, SNAP25, and syntaxin1. These proteins are required for vesicle priming and fusion. We generated an improved SNAP25-based SNARE COmplex Reporter (SCORE2) incorporating mCeruelan3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. This construct rescues vesicle fusion with properties indistinguishable from fusion in wild-type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR-FRET) imaging reveals a rapid FRET increase preceding individual fusion events by 65 ms. The experiments are performed under conditions of a steady-state cycle of docking, priming, and fusion, and the delay suggests that the FRET change reflects tight docking and priming of the vesicle, followed by fusion after ~65 ms. Given the absence of wt SNAP25, SCORE2 allows determination of the number of molecules at fusion sites and the number that changes conformation. The number of SNAP25 molecules changing conformation in the priming step increases with vesicle size and SNAP25 density in the plasma membrane and equals the number of copies present in the vesicle-plasma membrane contact zone. We estimate that in wt cells, 6 to 7 copies of SNAP25 change conformation during the priming step.

Single vesicle chemistry reveals partial release happens at the mechanical stress-induced exocytosis.

Neuronal activity can be modulated by mechanical stress in the central nervous system (CNS) in neurodegenerative diseases, for example Alzheimer's disease. However, the impact of mechanical stress on chemical signal transmission, especially the storage and release of neurotransmitter in neuron vesicles, has not been fully clarified. In this study, a nanotip conical carbon fiber microelectrode (CFME) and a disk CFME are placed in and on a cell, respectively. The nanotip conical CFME functions for both the mechanical stress and the quantification of transmitter storage in single vesicles, while the disk CFME is used to monitor the transmitter release during exocytosis induced by mechanical stress at the same cell. By comparing the vesicular transmitter storage with its release during mechanical stress-induced exocytosis at the same cell, we find the release ratio of transmitter in chromaffin cells varies from 27 % to 100 %, while for PC12 cells from 30 % to 100 %. Our results indicate that the exocytosis of cells responding to mechanical stress shows individual difference obviously, with a significant population exhibiting partial release mode. The variation of Ca2+ channels and mechanosensitive ion channels on cell membrane may both contribute to this variation. Our discovery not only shows mechanical stress can change the transmission of cellular chemical signals at the vesicle level, but also provides an important reference perspective for the study of nervous system regulation and nervous system diseases.

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

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

ADP-mediated Modulation of Intracellular Calcium Responses in Chromaffin Cells: The Role of Ectonucleoside Triphosphate Diphosphohydrolase 2 on Rat Adrenal Medulla Function.

The present study investigated the localization and the adenosine 5'-triphosphate (ATP)-degrading function of the plasma membrane-bound ecto-nucleotidase, ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2), in the rat adrenal medulla. The effect of ATP degradation product, adenosine 5'-diphosphate (ADP), on carbachol (CCh)-induced intracellular Ca2+ ([Ca2+]i) responses in adrenal chromaffin cells was examined using calcium imaging. NTPDase2-immunoreactive cells were distributed between chromaffin cells. NTPDase2-immunoreactive cells were immunoreactive for glial fibrillary acidic protein and S100B, suggesting that they were sustentacular cells. NTPDase2-immunoreactive cells surrounded chromaffin cells immunoreactive for vesicular nucleotide transporter and P2Y12 ADP-selective purinoceptors. In ATP bioluminescence assays using adrenal medullary slices, ATP was rapidly degraded and its degradation was attenuated by the NTPDase inhibitors sodium polyoxotungstate (POM-1) and 6-N, N-diethyl-d-ß,γ-dibromomethylene ATP (ARL67156). ADP inhibited CCh-induced [Ca2+]i increases of chromaffin cells in adrenal medullary slices. The inhibition of CCh-induced [Ca2+]i increases by ADP was blocked by the P2Y12 purinoceptor antagonist AZD1283. CCh-induced [Ca2+]i increases were also inhibited by the P2Y1, P2Y12, and P2Y13 purinoceptor agonist 2-methylthioadenosine diphosphate trisodium (2MeSADP), in combination with the P2Y1 purinoceptor antagonist MRS2179. These results suggest that sustentacular cells express NTPDase2 to degrade ATP released from adrenal chromaffin cells, and ADP modulates the excitability of chromaffin cells via P2Y12 purinoceptors to regulate catecholamine release during preganglionic sympathetic stimuli. (J Histochem Cytochem 72: 41-60, 2024).

Untapped Potential: Real-Time Measurements of Opioid Exocytosis at Single Cells.

The endogenous opioid system is commonly targeted in pain treatment, but the fundamental nature of neuropeptide release remains poorly understood due to a lack of methods for direct detection of specific opioid neuropeptides in situ. These peptides are concentrated in, and released from, large dense-core vesicles in chromaffin cells. Although catecholamine release from these neuroendocrine cells is well characterized, the direct quantification of opioid peptide exocytosis events has not previously been achieved. In this work, a planar carbon-fiber microelectrode served as a "postsynaptic" sensor for probing catecholamine and neuropeptide release dynamics via amperometric monitoring. A constant potential of 500 mV was employed for quantification of catecholamine release, and a higher potential of 1000 mV was used to drive oxidation of tyrosine, the N-terminal amino acid in the opioid neuropeptides released from chromaffin cells. By discriminating the results collected at the two potentials, the data reveal unique kinetics for these two neurochemical classes at the single-vesicle level. The amplitude of the peptidergic signals decreased with repeat stimulation, as the halfwidth of these signals simultaneously increased. By contrast, the amplitude of catecholamine release events increased with repeat stimulation, but the halfwidth of each event did not vary. The chromogranin dense core was identified as an important mechanistic handle by which separate classes of transmitter can be kinetically modulated when released from the same population of vesicles. Overall, the data provide unprecedented insight into key differences between catecholamine and opioid neuropeptide release from isolated chromaffin cells.

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

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

The aggressiveness of succinate dehydrogenase subunit B-deficient chromaffin cells is reduced when their bioelectrical properties are restored by glibenclamide.

Pheochromocytomas/paragangliomas (PPGLs) are neuroendocrine tumours, mostly resulting from mutations in predisposing genes. Mutations of succinate dehydrogenase (SDH) subunit B (SDHB) are associated with high probability of metastatic disease. Since bioelectrical properties and signalling in cancer are an emerging field, we investigated the metabolic, functional and electrophysiological characteristics in human succinate dehydrogenase subunit B (SDHB)-deficient pheochromocytoma cells. These cells exhibited reduced SDH function with elevated succinate-to-fumarate ratio and reduced intracellular ATP levels. The analysis of membrane passive properties revealed a more hyperpolarized membrane potential and a lower cell capacitance of SDHB-deficient cells compared to the parental ones. These bioelectrical changes were associated with reduced proliferation and adhesion capacity of SDHB-deficient cells. Only in SDHB-deficient cells, we also observed an increased amplitude of potassium currents suggesting an activation of ATP-sensitive potassium channels (KATP). Indeed, exposure of the SDHB-deficient cells to glibenclamide, a specific KATP inhibitor, or to ATP caused normalization of potassium current features and altered proliferation and adhesion. In this work, we show for the first time that reduced intracellular ATP levels in SDHB-deficient chromaffin cells impaired cell bioelectrical properties, which, in turn, are associated with an increased cell aggressiveness. Moreover, we first ever demonstrated that glibenclamide not only reduced the outward potassium currents in SDHB-deficient cells but increased their growth capacity, reduced their ability to migrate and shifted their phenotype towards one more similar to that of parental one.

Enhancement of muscarinic receptor-mediated excitation in spontaneously hypertensive rat adrenal medullary chromaffin cells.

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 M1 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.

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

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

Phospholipase C-ε defines a PACAP-stimulated pathway for secretion in the chromaffin cell.

Adrenomedullary chromaffin cells respond to splanchnic (sympathetic) nerve stimulation by releasing stress hormones into the circulation. The signal for hormone secretion is encoded in the neurotransmitters - especially acetylcholine (ACh) and pituitary adenylate cyclase activating polypeptide (PACAP) - that are released into the splanchnic-chromaffin cell synapse. However, functional differences in the effects of ACh and PACAP on the chromaffin cell secretory response are not well defined. Here, selective agonists of PACAP receptors or nicotinic and muscarinic acetylcholine receptors were applied to chromaffin cells. The major differences in the effects of these agents were not on exocytosis, per se, but rather on the steps upstream of exocytosis. In almost every respect, the properties of individual fusion events triggered by PACAP and cholinergic agonists were similar. On the other hand, the properties of the Ca2+ transients evoked by PACAP differed in several ways from those evoked by muscarinic and nicotinic receptor stimulation. A defining feature of the PACAP-stimulated secretory pathway was its dependence on signaling through exchange protein directly activated by cAMP (Epac) and PLCε. However, the absence of PLCε did not disrupt Ca2+ transients evoked by cholinergic agonists. Accordingly, inhibition of Epac activity did not disrupt secretion triggered by acetylcholine or specific agonists of muscarinic and nicotinic receptors. Thus, PACAP and acetylcholine stimulate chromaffin cell secretion via separate and independent pathways. This feature of stimulus-secretion coupling may be important for sustaining hormone release from the adrenal medulla under conditions associated with the sympathetic stress response.

Synaptotagmin-7 facilitates acetylcholine release in splanchnic nerve-chromaffin cell synapses during nerve activity.

Disturbances that threaten homeostasis elicit activation of the sympathetic nervous system (SNS) and the adrenal medulla. The effectors discharge as a unit to drive global and immediate changes in whole-body physiology. Descending sympathetic information is conveyed to the adrenal medulla via preganglionic splanchnic fibers. These fibers pass into the gland and synapse onto chromaffin cells, which synthesize, store, and secrete catecholamines and vasoactive peptides. While the importance of the sympatho-adrenal branch of the autonomic nervous system has been appreciated for many decades, the mechanisms underlying transmission between presynaptic splanchnic neurons and postsynaptic chromaffin cells have remained obscure. In contrast to chromaffin cells, which have enjoyed sustained attention as a model system for exocytosis, even the Ca2+ sensors that are expressed within splanchnic terminals have not yet been identified. This study shows that a ubiquitous Ca2+-binding protein, synaptotagmin-7 (Syt7), is expressed within the fibers that innervate the adrenal medulla, and that its absence can alter synaptic transmission in the preganglionic terminals of chromaffin cells. The prevailing impact in synapses that lack Syt7 is a decrease in synaptic strength and neuronal short-term plasticity. Evoked excitatory postsynaptic currents (EPSCs) in Syt7 KO preganglionic terminals are smaller in amplitude than in wild-type synapses stimulated in an identical manner. Splanchnic inputs also display robust short-term presynaptic facilitation, which is compromised in the absence of Syt7. These data reveal, for the first time, a role for any synaptotagmin at the splanchnic-chromaffin cell synapse. They also suggest that Syt7 has actions at synaptic terminals that are conserved across central and peripheral branches of the nervous system.

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

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

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

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

Two distinct pathways regulate chromaffin cell exocytosis.

JGP study reveals how the neurotransmitter PACAP induces a secretory response in chromaffin cells that differs from the one induced by acetylcholine.

Nerve growth factor causes epinephrine release dysfunction by regulating phenotype alterations and the function of adrenal medullary chromaffin cells in mice with allergic rhinitis.

The presence of allergic rhinitis (AR) is an increased risk factor for the occurrence of bronchial asthma (BA). Nerve growth factor (NGF), in addition to its key role in the development and differentiation of neurons, may also be an important inflammatory factor in AR and BA. However, the pathogenesis of the progression of AR to BA remains to be elucidated. The present study aimed to investigate the ability of NGF to mediate nasobronchial interactions and explore possible underlying molecular mechanisms. In the present study, an AR mouse model was established and histology of nasal mucosa tissue injury was determined. The level of phenylethanolamine N­methyl transferase in adrenal medulla was determined by immunofluorescence. Primary adrenal medullary chromaffin cells (AMCCs) were isolated and cultured from the adrenal medulla of mice. The expression levels of synaptophysin (SYP), STAT1, JAK1, p38 and ERK in NGF­treated and untreated AMCCs were detected by reverse­transcription­quantitative PCR and western blotting. The epinephrine (EPI) and norepinephrine (NE) concentrations were measured by ELISA. It was found that the expression of SYP in AMCCs was enhanced in the presence of NGF, whereas, the concentration of EPI decreased significantly under the same conditions. Furthermore, NGF mediated the phenotypic and functional changes of AMCCs, resulting in decreased EPI secretion via JAK1/STAT1, p38 and ERK signaling. In conclusion, these findings could provide novel evidence for the role of NGF in regulating neuroendocrine mechanisms.

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

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

Transmission Electron Microscopy: A Method for Studying the Adrenal Chromaffin Cells.

Transmission electron microscopy and the use of glutaraldehyde-osmium fixation allow to distinguish norepinephrine from epinephrine granules in the adrenochromaffin cells, a difficult distinction with histochemical methods if both types of granules are present in the same cell. Here we describe all the steps necessary to process the adrenochromaffin tissue for the transmission electron microscopy; this protocol is suitable for any kind of adrenal tissue, and personally we used it in mammals, reptiles, and amphibians.

Measurements of Calcium in Chromaffin Cell Organelles Using Targeted Aequorins.

The molecular mechanisms that mediate and regulate calcium (Ca2+) fluxes through the membranes of intracellular organelles play a key role in the generation and shaping of the local and global cytosolic Ca2+ signals triggering the process of regulated exocytosis in chromaffin cells. Beyond that role, intraorganellar Ca2+ homeostasis also regulates organelle-specific processes such as oxidative phosphorylation in mitochondria, maturation of secretory granules, or stress in the endoplasmic reticulum. In this chapter, we describe current methods to study mitochondrial, endoplasmic reticulum, and secretory vesicle calcium homeostasis in living chromaffin cells using engineered targeted aequorins.