Isolation and in vitro cultivation of adrenal cells from mice.

The adrenal gland consists of two tissues, cortex and medulla, united under one capsule. Adrenal stem/progenitor cells play a key role in development and homeostasis. Here, we describe a protocol for generating primary cultures of adrenal cells from mice. We describe techniques for separating the cortex and medulla, generating spheroid cultures containing stem- and progenitor cells, and for the differentiation into steroidogenic and chromaffin cells, respectively. This protocol enables analysis of various treatments before, during, or after differentiation. For complete details on the use and execution of this protocol, please refer to Rubin de Celis et al. (2015), Steenblock et al. (2018), and Werdermann et al. (2021).

Case Report: Irreversible Watery Diarrhea, Severe Metabolic Acidosis, Hypokalemia and Achloridria Syndrome Related to Vasoactive Intestinal Peptide Secreting Malignant Pheochromocytoma.

Background: Pheochromocytoma (PHEO) clinical manifestations generally mirror excessive catecholamines secretion; rarely the clinical picture may reflect secretion of other hormones. Watery diarrhea, hypokalemia and achlorhydria (WDHA) is a rare syndrome related to excessive secretion of vasoactive intestinal peptide (VIP). Clinical Case: A 73-year-old hypotensive man affected by adrenal PHEO presented with weight loss and watery diarrhea associated with hypokalemia, hyperchloremic metabolic acidosis (anion gap 15 mmol/l) and a negative urinary anion gap. Abdominal computed tomography scan showed a right adrenal PHEO, 8.1 cm in maximum diameter, with tracer uptake on 68GaDOTA-octreotate positron emission tomography. Metastasis in lumbar region and lung were present. Both chromogranin A and VIP levels were high (more than10 times the normal value) with slightly elevated urine normetanephrine and metanephrine excretion. Right adrenalectomy was performed and a somatostatin analogue therapy with lanreotide started. Immunostaining showed chromogranin A and VIP co-expression, with weak somatostatin-receptor-2A positivity. In two months, patient clinical conditions deteriorated with severe WDHA and multiple liver and lung metastasis. Metabolic acidosis and hypokalemia worsened, leading to hemodynamic shock and exitus. Conclusions: A rare case of WDHA syndrome caused by malignant VIP-secreting PHEO was diagnosed. High levels of circulating VIP were responsible of the rapidly evolving clinical picture with massive dehydration and weight loss along with severe hyperchloremic metabolic acidosis and hypokalemia due to the profuse untreatable diarrhea. The rescue treatment with lanreotide was unsuccessful because of the paucity of somatostatin-receptor-2A on VIP-secreting PHEO chromaffin cells.

Novel Purine Derivative ITH15004 Facilitates Exocytosis through a Mitochondrial Calcium-Mediated Mechanism.

Upon depolarization of chromaffin cells (CCs), a prompt release of catecholamines occurs. This event is triggered by a subplasmalemmal high-Ca2+ microdomain (HCMD) generated by Ca2+ entry through nearby voltage-activated calcium channels. HCMD is efficiently cleared by local mitochondria that avidly take up Ca2+ through their uniporter (MICU), then released back to the cytosol through mitochondrial Na+/Ca2+ exchanger (MNCX). We found that newly synthesized derivative ITH15004 facilitated the release of catecholamines triggered from high K+-depolarized bovine CCs. Such effect seemed to be due to regulation of mitochondrial Ca2+ circulation because: (i) FCCP-potentiated secretory responses decay was prevented by ITH15004; (ii) combination of FCCP and ITH15004 exerted additive secretion potentiation; (iii) such additive potentiation was dissipated by the MICU blocker ruthenium red (RR) or the MNCX blocker CGP37157 (CGP); (iv) combination of FCCP and ITH15004 produced both additive augmentation of cytosolic Ca2+ concentrations ([Ca2+]c) K+-challenged BCCs, and (v) non-inactivated [Ca2+]c transient when exposed to RR or CGP. On pharmacological grounds, data suggest that ITH15004 facilitates exocytosis by acting on mitochondria-controlled Ca2+ handling during K+ depolarization. These observations clearly show that ITH15004 is a novel pharmacological tool to study the role of mitochondria in the regulation of the bioenergetics and exocytosis in excitable cells.

Neuronal lineages derived from the nerve-associated Schwann cell precursors.

For a long time, neurogenic placodes and migratory neural crest cells were considered the immediate sources building neurons of peripheral nervous system. Recently, a number of discoveries revealed the existence of another progenitor type-a nerve-associated multipotent Schwann cell precursors (SCPs) building enteric and parasympathetic neurons as well as neuroendocrine chromaffin cells. SCPs are neural crest-derived and are similar to the crest cells by their markers and differentiation potential. Such similarities, but also considerable differences, raise many questions pertaining to the medical side, fundamental developmental biology and evolution. Here, we discuss the genesis of Schwann cell precursors, their role in building peripheral neural structures and ponder on their role in the origin in congenial diseases associated with peripheral nervous systems.

Bovine Chromaffin Cells: Culture and Fluorescence Assay for Secretion.

Over the last four decades, chromaffin cells originating from the adrenal medulla have been probably one of the most popular cell models to study neurosecretion at the molecular level. Accordingly, numerous seminal discoveries in the field, including the characterization of role of the cytoskeleton, fusogenic lipids, and soluble N-ethylmaleimide-sensitivefactor attachment protein receptor (SNARE) proteins, have been made using this model. In this chapter, we describe a standard method currently used to isolate and culture bovine chromaffin cells, and we illustrate a catecholamine secretion assay based on the successive transformation of adrenaline into adrenochrome and adrenolutine for fluorescence measurements. We also provide some guidelines for efficient cell recovery and for the use of this assay in the laboratory.

Single-Cell Characterization of Malignant Phenotypes and Developmental Trajectories of Adrenal Neuroblastoma.

Neuroblastoma (NB), which is a subtype of neural-crest-derived malignancy, is the most common extracranial solid tumor occurring in childhood. Despite extensive research, the underlying developmental origin of NB remains unclear. Using single-cell RNA sequencing, we generate transcriptomes of adrenal NB from 160,910 cells of 16 patients and transcriptomes of putative developmental cells of origin of NB from 12,103 cells of early human embryos and fetal adrenal glands at relatively late development stages. We find that most adrenal NB tumor cells transcriptionally mirror noradrenergic chromaffin cells. Malignant states also recapitulate the proliferation/differentiation status of chromaffin cells in the process of normal development. Our findings provide insight into developmental trajectories and cellular states underlying human initiation and progression of NB.

Paradoxical effects on voltage-gated Na+ conductance in adrenal chromaffin cells by twin vs single high intensity nanosecond electric pulses.

We previously reported that a single 5 ns high intensity electric pulse (NEP) caused an E-field-dependent decrease in peak inward voltage-gated Na+ current (INa) in isolated bovine adrenal chromaffin cells. This study explored the effects of a pair of 5 ns pulses on INa recorded in the same cell type, and how varying the E-field amplitude and interval between the pulses altered its response. Regardless of the E-field strength (5 to 10 MV/m), twin NEPs having interpulse intervals ≥ than 5 s caused the inhibition of TTX-sensitive INa to approximately double relative to that produced by a single pulse. However, reducing the interval from 1 s to 10 ms between twin NEPs at E-fields of 5 and 8 MV/m but not 10 MV/m decreased the magnitude of the additive inhibitory effect by the second pulse in a pair on INa. The enhanced inhibitory effects of twin vs single NEPs on INa were not due to a shift in the voltage-dependence of steady-state activation and inactivation but were associated with a reduction in maximal Na+ conductance. Paradoxically, reducing the interval between twin NEPs at 5 or 8 MV/m but not 10 MV/m led to a progressive interval-dependent recovery of INa, which after 9 min exceeded the level of INa reached following the application of a single NEP. Disrupting lipid rafts by depleting membrane cholesterol with methyl-ß-cyclodextrin enhanced the inhibitory effects of twin NEPs on INa and ablated the progressive recovery of this current at short twin pulse intervals, suggesting a complete dissociation of the inhibitory effects of twin NEPs on this current from their ability to stimulate its recovery. Our results suggest that in contrast to a single NEP, twin NEPs may influence membrane lipid rafts in a manner that enhances the trafficking of newly synthesized and/or recycling of endocytosed voltage-gated Na+ channels, thereby pointing to novel means to regulate ion channels in excitable cells.

The receptor tyrosine kinase EPHB6 regulates catecholamine exocytosis in adrenal gland chromaffin cells.

The erythropoietin-producing human hepatocellular receptor EPH receptor B6 (EPHB6) is a receptor tyrosine kinase that has been shown previously to control catecholamine synthesis in the adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent fashion. EPHB6 also has a role in regulating blood pressure, but several facets of this regulation remain unclear. Using amperometry recordings, we now found that catecholamine secretion by AGCCs is compromised in the absence of EPHB6. AGCCs from male knockout (KO) mice displayed reduced cortical F-actin disassembly, accompanied by decreased catecholamine secretion through exocytosis. This phenotype was not observed in AGCCs from female KO mice, suggesting that testosterone, but not estrogen, contributes to this phenotype. Of note, reverse signaling from EPHB6 to ephrin B1 (EFNB1) and a 7-amino acid-long segment in the EFNB1 intracellular tail were essential for the regulation of catecholamine secretion. Further downstream, the Ras homolog family member A (RHOA) and FYN proto-oncogene Src family tyrosine kinase (FYN)-proto-oncogene c-ABL-microtubule-associated monooxygenase calponin and LIM domain containing 1 (MICAL-1) pathways mediated the signaling from EFNB1 to the defective F-actin disassembly. We discuss the implications of EPHB6's effect on catecholamine exocytosis and secretion for blood pressure regulation.

8-Nitro-cGMP modulates exocytosis in adrenal chromaffin cells.

Nitric oxide (NO)-mediated production of cyclic guanosine 3',5'-monophosphate (cGMP) is a crucial signaling pathway that controls a wide array of neuronal functions, including exocytotic neurotransmitter release. A novel nitrated derivative of cGMP, 8-nitro-cGMP, not only activates cGMP-dependent protein kinase (PKG), but also has membrane permeability and redox activity to produce superoxide and S-guanylated protein. To date, no studies have addressed the effects of 8-nitro-cGMP on exocytotic kinetics. Here, we aimed to assess the 8-nitro-cGMP-mediated modulation of the depolarization-evoked catecholamine release from bovine chromaffin cells. 8-Nitro-cGMP was produced in bovine chromaffin cells dependent on NO donor. Amperometric analysis revealed that 8-nitro-cGMP modulated the kinetic parameters of secretory spikes from chromaffin cells, particularly decreased the speed of individual spikes, resulting in a reduced amperometric spike height, slope ß, and absolute value of slope γ. The modulatory effects were independent of the PKG signal and superoxide production. This is the first study to demonstrate that 8-nitro-cGMP modulates exocytosis and provide insights into a novel regulatory mechanism of exocytosis.

Schwann cell precursors contribute to skeletal formation during embryonic development in mice and zebrafish.

Immature multipotent embryonic peripheral glial cells, the Schwann cell precursors (SCPs), differentiate into melanocytes, parasympathetic neurons, chromaffin cells, and dental mesenchymal populations. Here, genetic lineage tracing revealed that, during murine embryonic development, some SCPs detach from nerve fibers to become mesenchymal cells, which differentiate further into chondrocytes and mature osteocytes. This occurred only during embryonic development, producing numerous craniofacial and trunk skeletal elements, without contributing to development of the appendicular skeleton. Formation of chondrocytes from SCPs also occurred in zebrafish, indicating evolutionary conservation. Our findings reveal multipotency of SCPs, providing a developmental link between the nervous system and skeleton.

RNA-seq of Isolated Chromaffin Cells Highlights the Role of Sex-Linked and Imprinted Genes in Adrenal Medulla Development.

Adrenal chromaffin cells and sympathetic neurons synthesize and release catecholamines, and both cell types are derived from neural crest precursors. However, they have different developmental histories, with sympathetic neurons derived directly from neural crest precursors while adrenal chromaffin cells arise from neural crest-derived cells that express Schwann cell markers. We have sought to identify the genes, including imprinted genes, which regulate the development of the two cell types in mice. We developed a method of separating the two cell types as early as E12.5, using differences in expression of enhanced yellow fluorescent protein driven from the tyrosine hydroxylase gene, and then used RNA sequencing to confirm the characteristic molecular signatures of the two cell types. We identified genes differentially expressed by adrenal chromaffin cells and sympathetic neurons. Deletion of a gene highly expressed by adrenal chromaffin cells, NIK-related kinase, a gene on the X-chromosome, results in reduced expression of adrenaline-synthesizing enzyme, phenyl-N-methyl transferase, by adrenal chromaffin cells and changes in cell cycle dynamics. Finally, many imprinted genes are up-regulated in chromaffin cells and may play key roles in their development.

Overexpression of P2X3 and P2X7 Receptors and TRPV1 Channels in Adrenomedullary Chromaffin Cells in a Rat Model of Neuropathic Pain.

We have tested the hypothesis that neuropathic pain acting as a stressor drives functional plasticity in the sympathoadrenal system. The relation between neuropathic pain and adrenal medulla function was studied with behavioral, immunohistochemical and electrophysiological techniques in rats subjected to chronic constriction injury of the sciatic nerve. In slices of the adrenal gland from neuropathic animals, we have evidenced increased cholinergic innervation and spontaneous synaptic activity at the splanchnic nerve⁻chromaffin cell junction. Likewise, adrenomedullary chromaffin cells displayed enlarged acetylcholine-evoked currents with greater sensitivity to α-conotoxin RgIA, a selective blocker of α9 subunit-containing nicotinic acetylcholine receptors, as well as increased exocytosis triggered by voltage-activated Ca2+ entry. Altogether, these adaptations are expected to facilitate catecholamine output into the bloodstream. Last, but most intriguing, functional and immunohistochemical data indicate that P2X3 and P2X7 purinergic receptors and transient receptor potential vanilloid-1 (TRPV1) channels are overexpressed in chromaffin cells from neuropathic animals. These latter observations are reminiscent of molecular changes characteristic of peripheral sensitization of nociceptors following the lesion of a peripheral nerve, and suggest that similar phenomena can occur in other tissues, potentially contributing to behavioral manifestations of neuropathic pain.

Expression pattern of delta-like 1 homolog in developing sympathetic neurons and chromaffin cells.

Delta-like 1 homolog (DLK1) is a member of the epidermal growth factor (EGF)-like family and an atypical notch ligand that is widely expressed during early mammalian development with putative functions in the regulation of cell differentiation and proliferation. During later stages of development, DLK1 is downregulated and becomes increasingly restricted to specific cell types, including several types of endocrine cells. DLK1 has been linked to various tumors and associated with tumor stem cell features. Sympathoadrenal precursors are neural crest derived cells that give rise to either sympathetic neurons of the autonomic nervous system or the endocrine chromaffin cells located in the adrenal medulla or extraadrenal positions. As these cells are the putative cellular origin of neuroblastoma, one of the most common malignant tumors in early childhood, their molecular characterization is of high clinical importance. In this study we have examined the precise spatiotemporal expression of DLK1 in developing sympathoadrenal cells. We show that DLK1 mRNA is highly expressed in early sympathetic neuron progenitors and that its expression depends on the presence of Phox2B. DLK1 expression becomes quickly restricted to a small subpopulation of cells in sympathetic ganglia, while virtually all chromaffin cells in the adrenal medulla and the Organ of Zuckerkandl still express high levels of DLK1 at late gestational stages.

RNA velocity of single cells.

RNA abundance is a powerful indicator of the state of individual cells. Single-cell RNA sequencing can reveal RNA abundance with high quantitative accuracy, sensitivity and throughput1. However, this approach captures only a static snapshot at a point in time, posing a challenge for the analysis of time-resolved phenomena such as embryogenesis or tissue regeneration. Here we show that RNA velocity-the time derivative of the gene expression state-can be directly estimated by distinguishing between unspliced and spliced mRNAs in common single-cell RNA sequencing protocols. RNA velocity is a high-dimensional vector that predicts the future state of individual cells on a timescale of hours. We validate its accuracy in the neural crest lineage, demonstrate its use on multiple published datasets and technical platforms, reveal the branching lineage tree of the developing mouse hippocampus, and examine the kinetics of transcription in human embryonic brain. We expect RNA velocity to greatly aid the analysis of developmental lineages and cellular dynamics, particularly in humans.

Modeling the influence of co-localized intracellular calcium stores on the secretory response of bovine chromaffin cells.

Catecholamines secretion from chromaffin cells is mediated by a Ca2+-dependent process in the submembrane space where the exocytotic machinery is located and high-Ca2+ microdomains (HCMDs) are formed by the coordinated activity of a functional triad composed of Ca2+ channels, endoplasmic reticulum (ER) and mitochondria. It has been observed experimentally that subpopulations of cortical mitochondria and ER associate to secretory sites in bovine chromaffin cells. Here, we study the effect of the geometrical distribution of the co-localized cortical organelles both in the formation of HCMDs in the vicinity of Ca2+ channels and on the secretory activity of bovine chromaffin cells in response to a single voltage pulse. Our simulations indicate that co-localized organelles have a dual role in the formation of HCMDs, having, on the one hand, an amplification effect due to the Ca2+-induced Ca2+-release mechanism from the ER and, on the other, acting as physical barriers to Ca2+ diffusion. In addition, our simulations suggest that the increased levels of Ca2+ in the microdomain enhances the secretion of the vesicles co-localized to the Ca2+ channels. As a whole, our results support the idea that the functional triads formed by Ca2+ channels, subplasmalemma ER and mitochondria have a positive effect on the secretion of catecholamines in bovine chromaffin cells.

Dual Antidepressant Duloxetine Blocks Nicotinic Receptor Currents, Calcium Signals and Exocytosis in Chromaffin Cells Stimulated with Acetylcholine.

The inhibition of nicotinic acetylcholine receptors (nAChRs) has been proposed as a potential strategy to develop new antidepressant drugs. This is based on the observation that antidepressants that selectively block noradrenaline (NA) or serotonin (5-HT) reuptake also inhibit nAChRs. Dual antidepressants blocking both NA and 5-HT reuptake were proposed to shorten the delay in exerting their clinical effects; whether duloxetine, a prototype of dual antidepressants, also blocks nAChRs is unknown. Here we explored this question in bovine chromaffin cells (BCCs) that express native α3, α5, and α7 nAChRs and in cell lines expressing human α7, α3ß4, or α4ß2 nAChRs. We have found that duloxetine fully blocked the acetylcholine (ACh)-elicited nicotinic currents in BCCs with an IC50 of 0.86 µM. Such blockade seemed to be noncompetitive, voltage dependent, and partially use dependent. The ACh-elicited membrane depolarization, the elevation of cytosolic calcium ([Ca2+]c), and catecholamine release in BCCs were also blocked by duloxetine. This blockade developed slowly, and the recovery of secretion was also slow and gradual. Duloxetine did not affect Na+ or Ca2+ channel currents neither the high-K+-elicited [Ca2+]c transients and secretion. Of interest was that in cell lines expressing human α7, α3ß4, and α4ß2 nAChRs, duloxetine blocked nicotinic currents with IC50 values of 0.1, 0.56, and 0.85 µM, respectively. Thus, in blocking α7 receptors, which are abundantly expressed in the brain, duloxetine exhibited approximately 10-fold to 100- fold higher potency with respect to reported IC50 values for various antidepressant drugs. This may contribute to the antidepressant effect of duloxetine.

[EPIGENETIC REGULATION OF ADRENAL CHROMAFFIN CELLS DEVELOPMENT (REVIEW)].

Chromaffin cells of adrenal medulla have traditionally been considered as modified sympathetic neurons. However, the results of recent studies indicate the need to revise this concept. The article reviews recent findings in origin and ontogeny of adrenal chromaffin cells and transcriptional and posttranscriptional regulation of developmental processes. The article summarizes data on transcriptional control of chromaffin cells proliferation and maturation and participation of microRNA in regulation of chromaffin and sympathetic neuronal phenotype gene expression.

Visualization of Membrane Pore in Live Cells Reveals a Dynamic-Pore Theory Governing Fusion and Endocytosis.

Fusion is thought to open a pore to release vesicular cargoes vital for many biological processes, including exocytosis, intracellular trafficking, fertilization, and viral entry. However, fusion pores have not been observed and thus proved in live cells. Its regulatory mechanisms and functions remain poorly understood. With super-resolution STED microscopy, we observed dynamic fusion pore behaviors in live (neuroendocrine) cells, including opening, expansion, constriction, and closure, where pore size may vary between 0 and 490 nm within 26 milliseconds to seconds (vesicle size: 180-720 nm). These pore dynamics crucially determine the efficiency of vesicular cargo release and vesicle retrieval. They are generated by competition between pore expansion and constriction. Pharmacology and mutation experiments suggest that expansion and constriction are mediated by F-actin-dependent membrane tension and calcium/dynamin, respectively. These findings provide the missing live-cell evidence, proving the fusion-pore hypothesis, and establish a live-cell dynamic-pore theory accounting for fusion, fission, and their regulation.

EPHB6 and testosterone in concert regulate epinephrine release by adrenal gland chromaffin cells.

Erythropoietin-producing human hepatocellular receptor (EPH) B6 (EPHB6) is a member of the receptor tyrosine kinase family. We previously demonstrated that EPHB6 knockout reduces catecholamine secretion in male but not female mice, and castration reverses this phenotype. We showed here that male EPHB6 knockout adrenal gland chromaffin cells presented reduced acetylcholine-triggered Ca2+ influx. Such reduction depended on the non-genomic effect of testosterone. Increased large conductance calcium-activated potassium channel current densities were recorded in adrenal gland chromaffin cells from male EPHB6 knockout mice but not from castrated knockout or female knockout mice. Blocking of the large conductance calcium-activated potassium channel in adrenal gland chromaffin cells from male knockout mice corrected their reduced Ca2+ influx. We conclude that the absence of EPHB6 and the presence of testosterone would lead to augmented large conductance calcium-activated potassium channel currents, which limit voltage-gated calcium channel opening in adrenal gland chromaffin cells. Consequently, acetylcholine-triggered Ca2+ influx is reduced, leading to lower catecholamine release in adrenal gland chromaffin cells from male knockout mice. This explains the reduced resting-state blood catecholamine levels, and hence the blood pressure, in male but not female EPHB6 knock mice. These findings have certain clinical implications.

Generation of Adrenal Chromaffin-like Cells from Human Pluripotent Stem Cells.

Adrenomedullary chromaffin cells are catecholamine (CA)-producing cells originating from trunk neural crest (NC) via sympathoadrenal progenitors (SAPs). We generated NC and SAPs from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in vitro via BMP2/FGF2 exposure, ascertained by qPCR and immunoexpression of SOX10, ASCL1, TFAP2α, and PHOX2B, and by fluorescence-activated cell sorting selection for p75NTR and GD2, and confirmed their trunk-like HOX gene expression. We showed that continuing BMP4 and curtailing FGF2 in vitro, augmented with corticosteroid mimetic, induced these cells to upregulate the chromaffin cell-specific marker PNMT and other CA synthesis and storage markers, and we demonstrated noradrenaline and adrenaline by Faglu and high-performance liquid chromatography. We showed these human cells' SAP-like property of migration and differentiation into cells expressing chromaffin cell markers by implanting them into avian embryos in vivo and in chorio-allantoic membrane grafts. These cells have the potential for investigating differentiation of human chromaffin cells and for modeling diseases involving this cell type.