Hif-2α programs oxygen chemosensitivity in chromaffin cells.

The study of transcription factors that determine specialized neuronal functions has provided invaluable insights into the physiology of the nervous system. Peripheral chemoreceptors are neurone-like electrophysiologically excitable cells that link the oxygen concentration of arterial blood to the neuronal control of breathing. In the adult, this oxygen chemosensitivity is exemplified by type I cells of the carotid body, and recent work has revealed one isoform of the hypoxia-inducible transcription factor (HIF), HIF-2α, as having a nonredundant role in the development and function of that organ. Here, we show that activation of HIF-2α, including isolated overexpression of HIF-2α but not HIF-1α, is sufficient to induce oxygen chemosensitivity in adult adrenal medulla. This phenotypic change in the adrenal medulla was associated with retention of extra-adrenal paraganglioma-like tissues resembling the fetal organ of Zuckerkandl, which also manifests oxygen chemosensitivity. Acquisition of chemosensitivity was associated with changes in the adrenal medullary expression of gene classes that are ordinarily characteristic of the carotid body, including G protein regulators and atypical subunits of mitochondrial cytochrome oxidase. Overall, the findings suggest that, at least in certain tissues, HIF-2α acts as a phenotypic driver for cells that display oxygen chemosensitivity, thus linking 2 major oxygen-sensing systems.

A PACAP-activated network for secretion requires coordination of Ca2+ influx and Ca2+ mobilization.

Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca2+ which are disrupted when Ca2+ influx through L-type channels is blocked or internal Ca2+ stores are depleted. PACAP liberates stored Ca2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca2+ mobilization to Ca2+ influx and supporting Ca2+-induced Ca2+-release. These Ca2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLCε) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ, is regulated by a signaling network that promotes sustained elevations in intracellular Ca2+ through multiple pathways.

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

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

AMPK inhibits voltage-gated calcium channel-current in rat chromaffin cells.

Metabolic changes are critical in the regulation of Ca2+ influx in central and peripheral neuroendocrine cells. To study the regulation of L-type Ca2+ channels by AMPK we used biochemical reagents and ATP/glucose-concentration manipulations in rat chromaffin cells. AICAR and Compound-C, at low concentration, significantly induce changes in L-type Ca2+ channel-current amplitude and voltage dependence. Remarkably, an overlasting decrease in the channel-current density can be induced by lowering the intracellular level of ATP. Accordingly, Ca2+ channel-current density gradually diminishes by decreasing the extracellular glucose concentration. By using immunofluorescence, a decrease in the expression of CaV1.2 is observed while decreasing extracellular glucose, suggesting that AMPK reduces the number of functional Ca2+ channels into the plasma membrane. Together, these results support for the first time the dependence of metabolic changes in the maintenance of Ca2+ channel-current by AMPK. They reveal a key step in Ca2+ influx in secretory cells.

Autocrine activation of P2X7 receptors mediates catecholamine secretion in chromaffin cells.

BACKGROUND AND PURPOSE: ATP is highly accumulated in secretory vesicles and secreted upon exocytosis from neurons and endocrine cells. In adrenal chromaffin granules, intraluminal ATP reaches concentrations over 100 mM. However, how these large amounts of ATP contribute to exocytosis has not been investigated. EXPERIMENTAL APPROACH: Exocytotic events in bovine and mouse adrenal chromaffin cells were measured with single cell amperometry. Cytosolic Ca2+ measurements were carried out in Fluo-4 loaded cells. Submembrane Ca2+ was examined in PC12 cells transfected with a membrane-tethered Ca2+ indicator Lck-GCaMP3. ATP release was measured using the luciferin/luciferase assay. Knockdown of P2X7 receptors was induced with short interfering RNA (siRNA). Direct Ca2+ influx through this receptor was measured using a P2X7 receptor-GCamp6 construct. KEY RESULTS: ATP induced exocytosis in chromaffin cells, whereas the ectonucleotidase apyrase reduced the release events induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP), high KCl, or ionomycin. The purinergic agonist BzATP also promoted a secretory response that was dependent on extracellular Ca2+. A740003, a P2X7 receptor antagonist, abolished secretory responses of these secretagogues. Exocytosis was also diminished in chromaffin cells when P2X7 receptors were silenced using siRNAs and in cells of P2X7 receptor knockout mice. In PC12 cells, DMPP induced ATP release, triggering Ca2+ influx through P2X7 receptors. Furthermore, BzATP, DMPP, and KCl allowed the formation of submembrane Ca2+ microdomains inhibited by A740003. CONCLUSION AND IMPLICATIONS: Autocrine activation of P2X7 receptors constitutes a crucial feedback system that amplifies the secretion of catecholamines in chromaffin cells by favouring submembrane Ca2+ microdomains.

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.

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

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

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).

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.

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.

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.

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.

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.

Methodologies for Detecting Quantal Exocytosis in Adrenal Chromaffin Cells Through Diamond-Based MEAs.

Diamond-based multiarray sensors are suitable to detect in real-time exocytosis and action potentials from cultured, spontaneously firing chromaffin cells, primary hippocampal neurons, and midbrain dopaminergic neurons. Here, we focus on how amperometric measurements of catecholamine release are performed on micrographitic diamond multiarrays (µG-D-MEAs) with high temporal and spatial resolution by 16 electrodes simultaneously.

Isolation and Purification of Chromaffin Granules from Adrenal Glands and Cultured Neuroendocrine Cells.

Chromaffin granules isolated from adrenal glands constitute a powerful experimental tool to the study of secretory vesicle components and their participation in fusion and docking processes, vesicle aggregation, and interactions with cytosolic components. Although it is possible to isolate and purify chromaffin granules from adrenal glands of different species, bovine adrenal glands are the most used tissue source due to its easy handling and the large amount of granules that can be obtained from this tissue. In this chapter, we describe an easy-to-use and short-term protocol for efficiently obtaining highly purified chromaffin granules from bovine adrenal medulla. We additionally include protocols to isolate granules from cultured bovine chromaffin cells and PC12 cells, as well as a section to obtain chromaffin granules from mouse adrenal glands.

PACAP and acetylcholine cause distinct Ca2+ signals and secretory responses in chromaffin cells.

The adrenomedullary chromaffin cell transduces chemical messages into outputs that regulate end organ function throughout the periphery. At least two important neurotransmitters are released by innervating preganglionic neurons to stimulate exocytosis in the chromaffin cell-acetylcholine (ACh) and pituitary adenylate cyclase activating polypeptide (PACAP). Although PACAP is widely acknowledged as an important secretagogue in this system, the pathway coupling PACAP stimulation to chromaffin cell secretion is poorly understood. The goal of this study is to address this knowledge gap. Here, it is shown that PACAP activates a Gαs-coupled pathway that must signal through phospholipase C ε (PLCε) to drive Ca2+ entry and exocytosis. PACAP stimulation causes a complex pattern of Ca2+ signals in chromaffin cells, leading to a sustained secretory response that is kinetically distinct from the form stimulated by ACh. Exocytosis caused by PACAP is associated with slower release of peptide cargo than exocytosis stimulated by ACh. Importantly, only the secretory response to PACAP, not ACh, is eliminated in cells lacking PLCε expression. The data show that ACh and PACAP, acting through distinct signaling pathways, enable nuanced and variable secretory outputs from chromaffin cells.

The antimicrobial peptides secreted by the chromaffin cells of the adrenal medulla link the neuroendocrine and immune systems: From basic to clinical studies.

The increasing resistance to antibiotic treatments highlights the need for the development of new antimicrobial agents. Antimicrobial peptides (AMPs) have been studied to be used in clinical settings for the treatment of infections. Endogenous AMPs represent the first line defense of the innate immune system against pathogens; they also positively interfere with infection-associated inflammation. Interestingly, AMPs influence numerous biological processes, such as the regulation of the microbiota, wound healing, the induction of adaptive immunity, the regulation of inflammation, and finally express anti-cancer and cytotoxic properties. Numerous peptides identified in chromaffin secretory granules from the adrenal medulla possess antimicrobial activity: they are released by chromaffin cells during stress situations by exocytosis via the activation of the hypothalamo-pituitary axis. The objective of the present review is to develop complete informations including (i) the biological characteristics of the AMPs produced after the natural processing of chromogranins A and B, proenkephalin-A and free ubiquitin, (ii) the design of innovative materials and (iii) the involvement of these AMPs in human diseases. Some peptides are elective biomarkers for critical care medicine, may play an important role in the protection of infections (alone, or in combination with others or antibiotics), in the prevention of nosocomial infections, in the regulation of intestinal mucosal dynamics and of inflammation. They could play an important role for medical implant functionalization, such as catheters, tracheal tubes or oral surgical devices, in order to prevent infections after implantation and to promote the healing of tissues.

LYTIC COCKTAIL ATTENUATES CATECHOLAMINE SURGE AFTER SEVERE BURNS BY BLOCKING HISTAMINE H1 RECEPTOR/PKA/CREB/TYROSINE HYDROXYLASE SIGNALING IN CHROMAFFIN CELLS.

ABSTRACT: Severe burns develop a catecholamine surge, inducing severe damage to the organism, raising the possibility of multisystem organ failure, and even death. The mechanisms of catecholamine surge have not been fully elucidated, and few strategies are generally acceptable to reduce catecholamine surge postburn. Thus, it is valuable to investigate the underlying mechanisms of catecholamine surge postburn to develop targeted interventions to attenuate it. We have found that the lytic cocktail alleviates the surge of catecholamine and organ injury after severe burn; however, the underlying mechanisms were still unclear. Moreover, the lytic cocktail has side effects, such as significant arterial hypotension and breathing depression, limiting its clinical application. This study aims to investigate the therapeutic mechanism of the lytic cocktail in regulating catecholamine levels postburn. We find that promethazine, a classic histamine H1 receptor blocker and a component of the lytic cocktail, can effectively reduce catecholamine surge and organ injury postburn. Our study confirms that blood histamine levels increase after severe burns. We find that histamine can amplify the catecholamine surge by elevating tyrosine hydroxylase expression and catecholamine synthesis in chromaffin cells through the histamine H1 receptor/Protein Kinase A /cAMP-response element binding protein signaling pathway. In summary, for the first time, we find that histamine plays a vital role in catecholamine surge postburn. We also confirm that the lytic cocktail effectively alleviates catecholamine surge and organ injury postburn through promethazine.

Serotonin limits generation of chromaffin cells during adrenal organ development.

Adrenal glands are the major organs releasing catecholamines and regulating our stress response. The mechanisms balancing generation of adrenergic chromaffin cells and protecting against neuroblastoma tumors are still enigmatic. Here we revealed that serotonin (5HT) controls the numbers of chromaffin cells by acting upon their immediate progenitor "bridge" cells via 5-hydroxytryptamine receptor 3A (HTR3A), and the aggressive HTR3Ahigh human neuroblastoma cell lines reduce proliferation in response to HTR3A-specific agonists. In embryos (in vivo), the physiological increase of 5HT caused a prolongation of the cell cycle in "bridge" progenitors leading to a smaller chromaffin population and changing the balance of hormones and behavioral patterns in adulthood. These behavioral effects and smaller adrenals were mirrored in the progeny of pregnant female mice subjected to experimental stress, suggesting a maternal-fetal link that controls developmental adaptations. Finally, these results corresponded to a size-distribution of adrenals found in wild rodents with different coping strategies.

Gambierol Blocks a K+ Current Fraction without Affecting Catecholamine Release in Rat Fetal Adrenomedullary Cultured Chromaffin Cells.

Gambierol inhibits voltage-gated K+ (KV) channels in various excitable and non-excitable cells. The purpose of this work was to study the effects of gambierol on single rat fetal (F19-F20) adrenomedullary cultured chromaffin cells. These excitable cells have different types of KV channels and release catecholamines. Perforated whole-cell voltage-clamp recordings revealed that gambierol (100 nM) blocked only a fraction of the total outward K+ current and slowed the kinetics of K+ current activation. The use of selective channel blockers disclosed that gambierol did not affect calcium-activated K+ (KCa) and ATP-sensitive K+ (KATP) channels. The gambierol concentration necessary to inhibit 50% of the K+ current-component sensitive to the polyether (IC50) was 5.8 nM. Simultaneous whole-cell current-clamp and single-cell amperometry recordings revealed that gambierol did not modify the membrane potential following 11s depolarizing current-steps, in both quiescent and active cells displaying repetitive firing of action potentials, and it did not increase the number of exocytotic catecholamine release events, with respect to controls. The subsequent addition of apamin and iberiotoxin, which selectively block the KCa channels, both depolarized the membrane and enhanced by 2.7 and 3.5-fold the exocytotic event frequency in quiescent and active cells, respectively. These results highlight the important modulatory role played by KCa channels in the control of exocytosis from fetal (F19-F20) adrenomedullary chromaffin cells.