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1.
J Gen Physiol ; 155(2)2023 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-36661929

RESUMO

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


Assuntos
Células Cromafins , Células Cromafins/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Exocitose/fisiologia
2.
Mol Med Rep ; 27(2)2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36601769

RESUMO

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.


Assuntos
Asma , Células Cromafins , Rinite Alérgica , Ratos , Animais , Camundongos , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Ratos Sprague-Dawley , Epinefrina/farmacologia , Asma/metabolismo , Rinite Alérgica/metabolismo , Células Cromafins/metabolismo , Fenótipo
3.
Proc Natl Acad Sci U S A ; 120(1): e2214897120, 2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36574702

RESUMO

During exocytosis, the fusion of secretory vesicle with plasma membrane forms a pore that regulates release of neurotransmitter and peptide. Heterogeneity of fusion pore behavior has been attributed to stochastic variation in a common exocytic mechanism, implying a lack of biological control. Using a fluorescent false neurotransmitter (FFN), we imaged dense core vesicle (DCV) exocytosis in primary mouse adrenal chromaffin cells by total internal reflection fluorescence microscopy at millisecond resolution and observed strikingly divergent modes of release, with fast events lasting <30 ms and slow events persisting for seconds. Dual imaging of slow events shows a delay in the entry of external dye relative to FFN release, suggesting exclusion by an extremely narrow pore <1 nm in diameter. Unbiased comprehensive analysis shows that the observed variation cannot be explained by stochasticity alone, but rather involves distinct mechanisms, revealing the bimodal nature of DCV exocytosis. Further, loss of calcium sensor synaptotagmin 7 increases the proportion of slow events without changing the intrinsic properties of either class, indicating the potential for independent regulation. The identification of two distinct mechanisms for release capable of independent regulation suggests a biological basis for the diversity of fusion pore behavior.


Assuntos
Células Cromafins , Vesículas de Núcleo Denso , Camundongos , Animais , Sinaptotagminas/metabolismo , Exocitose/fisiologia , Membrana Celular/metabolismo , Células Cromafins/metabolismo , Vesículas Secretórias/metabolismo , Fusão de Membrana/fisiologia , Cálcio/metabolismo
5.
J Gen Physiol ; 155(2)2023 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-36538657

RESUMO

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.


Assuntos
Células Cromafins , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Acetilcolina/farmacologia , Acetilcolina/metabolismo , Cálcio/metabolismo , Catecolaminas/metabolismo , Células Cromafins/metabolismo
6.
Bull Exp Biol Med ; 173(6): 783-786, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36322318

RESUMO

We analyzed the expression of the transcription factor Oct4 in the chromaffin cells during the postnatal development of the adrenal glands in rats. Single Oct4+-chromaffin cells with nuclear localization of the protein were found in the medulla, and their number increased in parallel with a decrease in the proliferative activity of cells. In postnatal development, 100% of chromaffin cells demonstrated high expression of tyrosine hydroxylase, which attested to their differentiation and functional activity. It was found that all Oct4+ cells were differentiated chromaffin cells. An increase in the pool of Oct4-expressing cells after the completion of organ growth suggests the formation of a potential source for the physiological and reparative regeneration of the adrenal medulla.


Assuntos
Medula Suprarrenal , Células Cromafins , Animais , Ratos , Glândulas Suprarrenais/metabolismo , Medula Suprarrenal/metabolismo , Diferenciação Celular , Células Cromafins/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo
7.
Front Endocrinol (Lausanne) ; 13: 1020000, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36237181

RESUMO

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


Assuntos
Células Cromafins , Glândulas Suprarrenais , Adrenérgicos , Animais , Diferenciação Celular/fisiologia , Feminino , Humanos , Camundongos , Norepinefrina , Oxigênio , Gravidez
8.
Front Immunol ; 13: 977175, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36090980

RESUMO

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.


Assuntos
Medula Suprarrenal , Células Cromafins , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos , Peptídeos Antimicrobianos , Humanos , Sistema Imunitário , Inflamação/tratamento farmacológico
9.
Shock ; 58(2): 158-168, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35953455

RESUMO

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.


Assuntos
Queimaduras , Células Cromafins , Queimaduras/tratamento farmacológico , Queimaduras/metabolismo , Catecolaminas , Células Cromafins/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Histamina/metabolismo , Histamina/farmacologia , Humanos , Prometazina/metabolismo , Receptores Histamínicos H1/metabolismo , Transdução de Sinais , Tirosina 3-Mono-Oxigenase/metabolismo
10.
Int J Mol Sci ; 23(16)2022 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-36012367

RESUMO

The heteromeric assembly of α3 and ß4 subunits of acetylcholine nicotinic receptors (nAChRs) seems to mediate the secretory response in bovine chromaffin cells. However, there is no information about the localization of these nAChRs in relationship with the secretory active zones in this cellular model. The present work presents the first evidence that, in fact, a population of these receptors is associated through the F-actin cytoskeleton with exocytotic machinery components, as detected by SNAP-25 labeling. Furthermore, we also prove that, upon stimulation, the probability to find α3ß4 nAChRs very close to exocytotic events increases with randomized distributions, thus substantiating the clear dynamic behavior of these receptors during the secretory process. Modeling on secretory dynamics and secretory component distributions supports the idea that α3ß4 nAChR cluster mobility could help with improving the efficiency of the secretory response of chromaffin cells. Our study is limited by the use of conventional confocal microscopy; in this sense, a strengthening to our conclusions could come from the use of super-resolution microscopy techniques in the near future.


Assuntos
Células Cromafins , Receptores Nicotínicos , Acetilcolina , Animais , Transporte Biológico , Bovinos , Células Cromafins/metabolismo , Antagonistas Nicotínicos , Receptores Nicotínicos/metabolismo
11.
Front Endocrinol (Lausanne) ; 13: 875865, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35795145

RESUMO

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


Assuntos
Medula Suprarrenal , Células Cromafins , Medula Suprarrenal/inervação , Medula Suprarrenal/metabolismo , Animais , Células Cromafins/metabolismo , Epinefrina , Mamíferos/metabolismo , Norepinefrina , Ratos , Nervos Esplâncnicos/metabolismo
12.
Anal Chem ; 94(27): 9548-9556, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35750055

RESUMO

For decades, carbon-fiber microelectrodes have been used in amperometric measurements of neurotransmitter release at a wide variety of cell types, providing a tremendous amount of valuable information on the mechanisms involved in dense-core vesicle fusion. The electroactive molecules that are released can be detected at the opposing microelectrode surface, allowing for precise quantification as well as detailed kinetic information on the stages of neurotransmitter release. However, it remains unclear how much of the catecholamine that is released into the artificial synapse escapes detection. This work examines two separate mechanisms by which released neurotransmitter goes undetected in a typical amperometric measurement. First, diffusional loss is assessed by monitoring exocytosis at single bovine chromaffin cells using carbon-fiber microelectrodes fabricated in a recessed (cavity) geometry. This creates a microsampling vial that minimizes diffusional loss of analyte prior to detection. More molecules were detected per exocytotic release event when using a recessed cavity sensor as compared to the conventional configuration. In addition, pharmacological inhibition of the norepinephrine transporter (NET), which serves to remove catecholamine from the extracellular space, increased both the size and the time course of individual amperometric events. Overall, this study characterizes distinct physical and biological mechanisms by which released neurotransmitter escapes detection at the opposing microelectrode surface, while also revealing an important role for the NET in "presynaptic" modulation of neurotransmitter release.


Assuntos
Células Cromafins , Exocitose , Animais , Fibra de Carbono , Catecolaminas/metabolismo , Bovinos , Células Cromafins/metabolismo , Exocitose/fisiologia , Microeletrodos , Neurotransmissores/metabolismo
13.
Nat Commun ; 13(1): 2901, 2022 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-35614045

RESUMO

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.


Assuntos
Células Cromafins , Neuroblastoma , Glândulas Suprarrenais/metabolismo , Animais , Catecolaminas/metabolismo , Células Cromafins/metabolismo , Feminino , Camundongos , Neuroblastoma/metabolismo , Gravidez , Serotonina/metabolismo
14.
Cell Calcium ; 105: 102606, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35636152

RESUMO

Exocytosis operates through two distinct modes. Full-fusion leads to rapid expulsion of the entire content of a vesicle; kiss-and-run leads to slow and partial expulsion. These two modes have important biological consequences for endocrine regulation and synaptic transmission. Amperometry recordings of catecholamine release from chromaffin cells reveal single-vesicle fusion events corresponding to both of these modes, but classification is often difficult. This study introduces a new method of analyzing amperometry data to improve this classification. The ratio of the average amplitude to the peak amplitude differs between full-fusion and kiss-and-run, and the probability distribution of this ratio is well fitted by a double-Gaussian. Kiss-and-run events identified by this method have fusion pores with kinetic properties different from pores associated with full-fusion. They have slower transition rates and lifetime distributions indicative of irreversible transitions. The total-charge of an amperometric spike is expected to scale with vesicle volume during a full-fusion event. The cube root of this quantity should therefore scale with diameter, but the distribution of this quantity differs from the distribution of vesicle diameter seen in the electron microscope. Fusion pore lifetimes associated with full-fusion depend on vesicle size, and this makes the choice of mode size dependent. The fusion pore thus bifurcates after opening, and vesicle size influences this choice. The secretory vesicle protein synaptophysin influences the size dependence of fusion pore lifetime and the choice of release mode. Incorporating vesicle size into an analysis of release mode reconciled the kinetics of fusion pores, as well as the distributions of vesicle diameter and catecholamine content. Thus, the initial fusion pore emerges as a critical focus in endocrine regulation. By modulating the size-dependence of the mode of exocytosis, changes in the molecular makeup of the exocytotic apparatus can impact the shape and size of an amperometric event, and the speed and composition of secretion.


Assuntos
Células Cromafins , Catecolaminas/metabolismo , Células Cromafins/metabolismo , Exocitose/fisiologia , Fusão de Membrana/fisiologia , Vesículas Secretórias/metabolismo
15.
Endocrinology ; 163(7)2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35595517

RESUMO

During fasting, increased sympathoadrenal activity leads to epinephrine release and multiple forms of plasticity within the adrenal medulla including an increase in the strength of the preganglionic → chromaffin cell synapse and elevated levels of agouti-related peptide (AgRP), a peptidergic cotransmitter in chromaffin cells. Although these changes contribute to the sympathetic response, how fasting evokes this plasticity is not known. Here we report these effects involve activation of GPR109A (HCAR2). The endogenous agonist of this G protein-coupled receptor is ß-hydroxybutyrate, a ketone body whose levels rise during fasting. In wild-type animals, 24-hour fasting increased AgRP-ir in adrenal chromaffin cells but this effect was absent in GPR109A knockout mice. GPR109A agonists increased AgRP-ir in isolated chromaffin cells through a GPR109A- and pertussis toxin-sensitive pathway. Incubation of adrenal slices in nicotinic acid, a GPR109A agonist, mimicked the fasting-induced increase in the strength of the preganglionic → chromaffin cell synapse. Finally, reverse transcription polymerase chain reaction experiments confirmed the mouse adrenal medulla contains GPR109A messenger RNA. These results are consistent with the activation of a GPR109A signaling pathway located within the adrenal gland. Because fasting evokes epinephrine release, which stimulates lipolysis and the production of ß-hydroxybutyrate, our results indicate that chromaffin cells are components of an autonomic-adipose-hepatic feedback circuit. Coupling a change in adrenal physiology to a metabolite whose levels rise during fasting is presumably an efficient way to coordinate the homeostatic response to food deprivation.


Assuntos
Ácido 3-Hidroxibutírico , Medula Suprarrenal , Células Cromafins , Jejum , Receptores Acoplados a Proteínas G , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/farmacologia , Medula Suprarrenal/citologia , Medula Suprarrenal/metabolismo , Proteína Relacionada com Agouti/metabolismo , Animais , Plasticidade Celular , Células Cromafins/metabolismo , Epinefrina/metabolismo , Jejum/metabolismo , Camundongos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo
16.
STAR Protoc ; 3(2): 101404, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35600934

RESUMO

Real-time confocal and super-resolution imaging reveals membrane dynamics of exo- and endocytosis, including hemi-fusion, fusion pore opening, expansion, constriction, closure (kiss-and-run), fused-vesicle shrinking (shrink fusion), and flat membrane transition to vesicles via intermediate Λ- and Ω-shape structures. Here, we describe a protocol for imaging these membrane dynamics, including primary culture of bovine adrenal chromaffin cells, fluorescent probe application, patch-clamp to deliver depolarization and evoke exo- and endocytosis, electron microscopy (EM), and real-time confocal and stimulated emission depletion (STED) microscopy. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2016), Shin et al. (2018), and Shin et al. (2021).


Assuntos
Células Cromafins , Fusão de Membrana , Animais , Bovinos , Endocitose , Microscopia/métodos , Vesículas Secretórias
17.
J Vis Exp ; (181)2022 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-35377362

RESUMO

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated Ω-shape profile until it merges completely at the plasma membrane. To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C δ (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.


Assuntos
Células Cromafins , Fusão de Membrana , Animais , Bovinos , Membrana Celular/metabolismo , Exocitose , Microscopia Confocal
18.
J Histochem Cytochem ; 70(5): 335-356, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35400231

RESUMO

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


Assuntos
Neoplasias das Glândulas Suprarrenais , Células Cromafins , Feocromocitoma , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/patologia , Neoplasias das Glândulas Suprarrenais/veterinária , Animais , Células Cromafins/metabolismo , Células Cromafins/patologia , Cromograninas/metabolismo , Cães , Humanos , Feocromocitoma/metabolismo , Feocromocitoma/patologia , Feocromocitoma/veterinária , Secretogranina II/metabolismo
19.
Toxins (Basel) ; 14(4)2022 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-35448863

RESUMO

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.


Assuntos
Células Cromafins , Ciguatoxinas , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/farmacologia , Catecolaminas/farmacologia , Células Cultivadas , Ciguatoxinas/farmacologia , Potássio , Ratos
20.
Arch Biochem Biophys ; 723: 109252, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35436445

RESUMO

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


Assuntos
Células Cromafins , Canais de Cátion TRPM , Animais , Cátions/metabolismo , Bovinos , Células Cromafins/metabolismo , Potenciais da Membrana , RNA Mensageiro/metabolismo , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPM/metabolismo , Wortmanina/metabolismo , Wortmanina/farmacologia
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