Modulating Ca2+ influx into adrenal chromaffin cells with short-duration nanosecond electric pulses.

Isolated bovine adrenal chromaffin cells exposed to single 2-, 4-, or 5-ns pulses undergo a rapid, transient rise in intracellular Ca2+ mediated by Ca2+ entry via voltage-gated Ca2+ channels (VGCCs), mimicking the activation of these cells in vivo by acetylcholine. However, pulse durations 150 ns or longer elicit larger amplitude and longer-lived Ca2+ responses due to Ca2+ influx via both VGCCs and a yet to be identified plasma membrane pathway(s). To further our understanding of the differential effects of ultrashort versus longer pulse durations on Ca2+ influx, chromaffin cells were loaded with calcium green-1 and exposed to single 3-, 5-, 11-, 25-, or 50-ns pulses applied at their respective Ca2+ activation threshold electric fields. Increasing pulse duration from 3 or 5 ns to only 11 ns was sufficient to elicit increased amplitude and longer-lived Ca2+ responses in the majority of cells, a trend that continued as pulse duration increased to 50 ns. The amplification of Ca2+ responses was not the result of Ca2+ release from intracellular stores and was accompanied by a decreased effectiveness of VGCC inhibitors to block the responses and a reduced reliance on extracellular Na+ and membrane depolarization to evoke the responses. Inhibitors of pannexin channels, P2X receptors, or non-selective cation channels failed to attenuate 50-ns-elicited Ca2+ responses, ruling out these Ca2+-permeable channels as secondary Ca2+ entry pathways. Analytical calculations and numerical modeling suggest that the parameter that best determines the response of chromaffin cells to increasing pulse durations is the time the membrane charges to its peak voltage. These results highlight the pronounced sensitivity of a neuroendocrine cell to pulse durations differing by only tens of nanoseconds, which has important implications for the future development of nanosecond pulse technologies enabling electrostimulation applications for spatially focused and graded in vivo neuromodulation.

Evolution of a novel adrenal cell type that promotes parental care.

Cell types with specialized functions fundamentally regulate animal behaviour, and yet the genetic mechanisms that underlie the emergence of novel cell types and their consequences for behaviour are not well understood1. Here we show that the monogamous oldfield mouse (Peromyscus polionotus) has recently evolved a novel cell type in the adrenal gland that expresses the enzyme AKR1C18, which converts progesterone into 20α-hydroxyprogesterone. We then demonstrate that 20α-hydroxyprogesterone is more abundant in oldfield mice, where it induces monogamous-typical parental behaviours, than in the closely related promiscuous deer mice (Peromyscus maniculatus). Using quantitative trait locus mapping in a cross between these species, we ultimately find interspecific genetic variation that drives expression of the nuclear protein GADD45A and the glycoprotein tenascin N, which contribute to the emergence and function of this cell type in oldfield mice. Our results provide an example by which the recent evolution of a new cell type in a gland outside the brain contributes to the evolution of social behaviour.

Munc18-dependent and -independent clustering of syntaxin in the plasma membrane of cultured endocrine cells.

Syntaxin helps in catalyzing membrane fusion during exocytosis. It also forms clusters in the plasma membrane, where both its transmembrane and SNARE domains are thought to homo-oligomerize. To study syntaxin clustering in live PC12 cells, we labeled granules with neuropeptide-Y-mCherry and syntaxin clusters with syntaxin-1a green fluorescent protein (GFP). Abundant clusters appeared under total internal reflection (TIRF) illumination, and some of them associated with granules ("on-granule clusters"). Syntaxin-1a-GFP or its mutants were expressed at low levels and competed with an excess of endogenous syntaxin for inclusion into clusters. On-granule inclusion was diminished by mutations known to inhibit binding to Munc18-1 in vitro. Knock-down of Munc18-1 revealed Munc18-dependent and -independent on-granule clustering. Clustering was inhibited by mutations expected to break salt bridges between syntaxin's Hb and SNARE domains and was rescued by additional mutations expected to restore them. Most likely, syntaxin is in a closed conformation when it clusters on granules, and its SNARE and Hb domains approach to within atomic distances. Pairwise replacements of Munc18-contacting residues with alanines had only modest effects, except that the pair R114A/I115A essentially abolished on-granule clustering. In summary, an on-granule cluster arises from the specific interaction between a granule and a dense cluster of syntaxin-Munc18-1 complexes. Off-granule clusters, by contrast, were resistant to even the strongest mutations we tried and required neither Munc18-1 nor the presence of a SNARE domain. They may well form through the nonstoichiometric interactions with membrane lipids that others have observed in cell-free systems.

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).

Progressive Mitochondrial SOD1G93A Accumulation Causes Severe Structural, Metabolic and Functional Aberrations through OPA1 Down-Regulation in a Mouse Model of Amyotrophic Lateral Sclerosis.

In recent years, the "non-autonomous motor neuron death" hypothesis has become more consolidated behind amyotrophic lateral sclerosis (ALS). It postulates that cells other than motor neurons participate in the pathology. In fact, the involvement of the autonomic nervous system is fundamental since patients die of sudden death when they become unable to compensate for cardiorespiratory arrest. Mitochondria are thought to play a fundamental role in the physiopathology of ALS, as they are compromised in multiple ALS models in different cell types, and it also occurs in other neurodegenerative diseases. Our study aimed to uncover mitochondrial alterations in the sympathoadrenal system of a mouse model of ALS, from a structural, bioenergetic and functional perspective during disease instauration. We studied the adrenal chromaffin cell from mutant SOD1G93A mouse at pre-symptomatic and symptomatic stages. The mitochondrial accumulation of the mutated SOD1G93A protein and the down-regulation of optic atrophy protein-1 (OPA1) provoke mitochondrial ultrastructure alterations prior to the onset of clinical symptoms. These changes affect mitochondrial fusion dynamics, triggering mitochondrial maturation impairment and cristae swelling, with increased size of cristae junctions. The functional consequences are a loss of mitochondrial membrane potential and changes in the bioenergetics profile, with reduced maximal respiration and spare respiratory capacity of mitochondria, as well as enhanced production of reactive oxygen species. This study identifies mitochondrial dynamics regulator OPA1 as an interesting therapeutic target in ALS. Additionally, our findings in the adrenal medulla gland from presymptomatic stages highlight the relevance of sympathetic impairment in this disease. Specifically, we show new SOD1G93A toxicity pathways affecting cellular energy metabolism in non-motor neurons, which offer a possible link between cell specific metabolic phenotype and the progression of ALS.

Histopathological Analysis of Adrenal Glands after Simian Varicella Virus Infection.

Latent varicella zoster virus (VZV) has been detected in human adrenal glands, raising the possibility of virus-induced adrenal damage and dysfunction during primary infection or reactivation. Rare cases of bilateral adrenal hemorrhage and insufficiency associated with VZV reactivation have been reported. Since there is no animal model for VZV infection of adrenal glands, we obtained adrenal glands from two non-human primates (NHPs) that spontaneously developed varicella from primary simian varicella virus (SVV) infection, the NHP VZV homolog. Histological and immunohistochemical analysis revealed SVV antigen and DNA in the adrenal medulla and cortex of both animals. Adrenal glands were observed to have Cowdry A inclusion bodies, cellular necrosis, multiple areas of hemorrhage, and varying amounts of polymorphonuclear cells. No specific association of SVV antigen with ßIII-tubulin-positive nerve fibers was found. Overall, we found that SVV can productively infect NHP adrenal glands, and is associated with inflammation, hemorrhage, and cell death. These findings suggest that further studies are warranted to examine the contribution of VZV infection to human adrenal disease. This study also suggests that VZV infection may present itself as acute adrenal dysfunction with "long-hauler" symptoms of fatigue, weakness, myalgias/arthralgias, and hypotension.

Expression of Transcription Factor CREM in Human Tissues.

Cyclic AMP element modulator (CREM) is a transcription factor best known for its intricate involvement in spermatogenesis. The CREM gene encodes for multiple protein isoforms, which can enhance or repress transcription of target genes. Recent studies have identified fusion genes, with CREM as a partner gene in many neoplastic diseases. EWSR1-CREM fusion genes have been found in several mesenchymal tumors and in salivary gland carcinoma. These genes encode fusion proteins that include the C-terminal DNA-binding domain of CREM. We used a transcriptomic approach and immunohistochemistry to study the expression of CREM isoforms that include DNA-binding domains across human tissues. We found that CREM protein is widely expressed in almost all normal human tissues. A transcriptomic analysis of normal tissues and cancer showed that transcription of CREM can be altered in tumors, suggesting that also wild-type CREM may be involved in cancer biology. The wide expression of CREM protein in normal human tissues and cancer may limit the utility of immunohistochemistry for identification of tumors with CREM fusions.

Single-cell transcriptomes reveal characteristic features of cell types within the human adrenal microenvironment.

Various cells within the adrenal microenvironment are important in maintaining the body homeostasis. However, our understanding of adrenal disease pathogenesis is limited by an incomplete molecular characterization of the cell types responsible for the organ's multiple homeostatic functions. We report a cellular landscape of the human adrenal gland using single-cell RNA sequencing. We reveal characteristic features of cell types within the human adrenal microenvironment and found immune activation of nonimmune cells in the adrenal endothelial cells. We also reveal that abundant immune cells occupied a lot of space in adrenal gland. Additionally, Sex-related diversity in the adrenocortical cells and different gene expression profiles between the left and right adrenal gland are also observed at single-cell resolution. Together, at single-cell resolution, the transcriptomic map presents a comprehensive view of the human adrenal gland, which serves as a fundamental baseline description of this organ and paves a way for the further studies of adrenal diseases.

5 ns electric pulses induce Ca2+-dependent exocytotic release of catecholamine from adrenal chromaffin cells.

Previously we reported that adrenal chromaffin cells exposed to a 5 ns, 5 MV/m pulse release the catecholamines norepinephrine (NE) and epinephrine (EPI) in a Ca2+-dependent manner. Here we determined that NE and EPI release increased with pulse number (one versus five and ten pulses at 1 Hz), established that release occurs by exocytosis, and characterized the exocytotic response in real-time. Evidence of an exocytotic mechanism was the appearance of dopamine-ß-hydroxylase on the plasma membrane, and the demonstration by total internal reflection fluorescence microscopy studies that a train of five or ten pulses at 1 Hz triggered the release of the fluorescent dye acridine orange from secretory granules. Release events were Ca2+-dependent, longer-lived relative to those evoked by nicotinic receptor stimulation, and occurred with a delay of several seconds despite an immediate rise in Ca2+. In complementary studies, cells labeled with the plasma membrane fluorescent dye FM 1-43 and exposed to a train of ten pulses at 1 Hz underwent Ca2+-dependent increases in FM 1-43 fluorescence indicative of granule fusion with the plasma membrane due to exocytosis. These results demonstrate the effectiveness of ultrashort electric pulses for stimulating catecholamine release, signifying their promise as a novel electrostimulation modality for neurosecretion.

GWAS of allometric body-shape indices in UK Biobank identifies loci suggesting associations with morphogenesis, organogenesis, adrenal cell renewal and cancer.

Genetic studies have examined body-shape measures adjusted for body mass index (BMI), while allometric indices are additionally adjusted for height. We performed the first genome-wide association study of A Body Shape Index (ABSI), Hip Index (HI) and the new Waist-to-Hip Index and compared these with traditional indices, using data from the UK Biobank Resource for 219,872 women and 186,825 men with white British ancestry and Bayesian linear mixed-models (BOLT-LMM). One to two thirds of the loci identified for allometric body-shape indices were novel. Most prominent was rs72959041 variant in RSPO3 gene, expressed in visceral adipose tissue and regulating adrenal cell renewal. Highly ranked were genes related to morphogenesis and organogenesis, previously additionally linked to cancer development and progression. Genetic associations were fewer in men compared to women. Prominent region-specific associations showed variants in loci VEGFA and HMGA1 for ABSI and KLF14 for HI in women, and C5orf67 and HOXC4/5 for ABSI and RSPO3, VEGFA and SLC30A10 for HI in men. Although more variants were associated with waist and hip circumference adjusted for BMI compared to ABSI and HI, associations with height had previously been reported for many of the additional variants, illustrating the importance of adjusting correctly for height.

Loss of CREB Coactivator CRTC1 in SF1 Cells Leads to Hyperphagia and Obesity by High-fat Diet But Not Normal Chow Diet.

Cyclic adenosine monophosphate responsive element-binding protein-1-regulated transcription coactivator-1 (CRTC1) is a cytoplasmic coactivator that translocates to the nucleus in response to cyclic adenosine monophosphate. Whole-body knockdown of Crtc1 causes obesity, resulting in increased food intake and reduced energy expenditure. CRTC1 is highly expressed in the brain; therefore, it might play an important role in energy metabolism via the neuronal pathway. However, the precise mechanism by which CRTC1 regulates energy metabolism remains unknown. Here, we showed that mice lacking CRTC1, specifically in steroidogenic factor-1 expressing cells (SF1 cells), were sensitive to high-fat diet (HFD)-induced obesity, exhibiting hyperphagia and increased body weight gain. The loss of CRTC1 in SF1 cells impaired glucose metabolism. Unlike whole-body CRTC1 knockout mice, SF1 cell-specific CRTC1 deletion did not affect body weight gain or food intake in normal chow feeding. Thus, CRTC1 in SF1 cells is required for normal appetite regulation in HFD-fed mice. CRTC1 is primarily expressed in the brain. Within the hypothalamus, which plays an important role for appetite regulation, SF1 cells are only found in ventromedial hypothalamus. RNA sequencing analysis of microdissected ventromedial hypothalamus samples revealed that the loss of CRTC1 significantly changed the expression levels of certain genes. Our results revealed the important protective role of CRTC1 in SF1 cells against dietary metabolic imbalance.

Adrenal gland fine needle aspiration: a multi-institutional analysis of 139 cases.

INTRODUCTION: Adrenal gland lesions span a range of entities from benign and malignant primary neoplasms to metastatic tumors. Fine-needle aspiration (FNA) provides a minimally invasive diagnostic tool to stage patients with known malignancy and procure material for molecular testing. This study characterizes the clinicopathologic associations of patients with adrenal gland FNA from 2 large hospitals. MATERIALS AND METHODS: FNAs were identified by query of electronic medical record from 2002-2019. Clinical and pathological information was collated and correlated with corresponding surgical diagnosis when available. RESULTS: Of 139 cases, the majority (n = 127, 91%) were adequate computed tomography-guided FNAs and included the following diagnoses: positive for malignancy (n = 77, 55%), negative for malignancy (n = 32, 23%), neoplastic cells present (n = 16, 12%), nondiagnostic (n = 12, 9%), atypical (n = 1, 1%), and suspicious for carcinoma (n = 1, 1%). The majority (94%, n = 72 of 77) of malignancies were metastatic tumors, most frequently carcinoma (n = 53 of 72, 74%), followed by melanoma (n = 11 of 72, 15%), lymphoma (n = 4 of 72, 6%), and sarcoma (n = 4 of 72, 6%). Metastatic carcinomas included lung (n = 21 of 72, 29%), genitourinary (n = 12 of 72, 17%), and hepatobiliary or gastrointestinal tract (n = 11 of 72, 15%) primaries. Primary adrenal neoplasms (n = 23) included adenomas (n = 11 of 23, 48%), pheochromocytomas (n = 4 of 23, 17%), and myelolipomas (n = 3 of 23, 13%). Thirty-two patients with metastases died of disease after median follow-up of 8 months. CONCLUSIONS: High specimen adequacy (n = 127, 91%) and low indeterminate rates (n = 2, 2%) are achieved with adrenal FNA. Most aspirated lesions represent metastases from primary lung carcinomas, but other primary sites including those below the diaphragm are part of the diagnostic differential. Adrenal metastasis was associated with a poor prognosis, with median survival of 8 months.

Establishment of a Novel Human Fetal Adrenal Culture Model that Supports de Novo and Manipulated Steroidogenesis.

CONTEXT: Disorders affecting adrenal steroidogenesis promote an imbalance in the normally tightly controlled secretion of mineralocorticoids, glucocorticoids, and androgens. This may lead to differences/disorders of sex development in the fetus, as seen in virilized girls with congenital adrenal hyperplasia (CAH). Despite the important endocrine function of human fetal adrenals, neither normal nor dysregulated adrenal steroidogenesis is understood in detail. OBJECTIVE: Due to significant differences in adrenal steroidogenesis between human and model species (except higher primates), we aimed to establish a human fetal adrenal model that enables examination of both de novo and manipulated adrenal steroidogenesis. DESIGN AND SETTING: Human adrenal tissue from 54 1st trimester fetuses were cultured ex vivo as intact tissue fragments for 7 or 14 days. MAIN OUTCOME MEASURES: Model validation included examination of postculture tissue morphology, viability, apoptosis, and quantification of steroid hormones secreted to the culture media measured by liquid chromatography-tandem mass spectrometry. RESULTS: The culture approach maintained cell viability, preserved cell populations of all fetal adrenal zones, and recapitulated de novo adrenal steroidogenesis based on continued secretion of steroidogenic intermediates, glucocorticoids, and androgens. Adrenocorticotropic hormone and ketoconazole treatment of ex vivo cultured human fetal adrenal tissue resulted in the stimulation of steroidogenesis and inhibition of androgen secretion, respectively, demonstrating a treatment-specific response. CONCLUSIONS: Together, these data indicate that ex vivo culture of human fetal adrenal tissue constitutes a novel approach to investigate local effects of pharmaceutical exposures or emerging therapeutic options targeting imbalanced steroidogenesis in adrenal disorders, including CAH.

Electro-acupuncture regulates glucose metabolism in chronic stress model rats.

Studies have shown that acupuncture is very effective in treating chronic stress depression. However, little is known about the therapeutic mechanism of electro-acupuncture. Metabolomics, on the other hand, is a technology that determines the metabolic changes of organisms caused by various interventions as a whole and is related to the overall effect of electro-acupuncture (EA). 1HNMR, serum sample analysis, and histopathology and molecular biology analysis were used to evaluate the effects of EA. The results show that electro-acupuncture points can regulate the heat pain threshold of chronic stress model rats and change the morphology of adrenal cortex cells Structure, and regulate the contents of corticotropin-releasing hormone, Corticosterone (CORT), glucose, alanine and valine in the samples. These findings help to clarify the therapeutic mechanism of electro-acupuncture on heterologous chronic stress model rats. The effect of electro-acupuncture on improving chronic stress is likely to be achieved by regulating glucose metabolism, which can provide a reference for clinical acupuncture treatment of chronic stress depression.

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 Potential Role of Aldosterone-Producing Cell Clusters in Adrenal Disease.

Primary aldosteronism (PA) is the most common cause of secondary hypertension. The hallmark of PA is adrenal production of aldosterone under suppressed renin conditions. PA subtypes include adrenal unilateral and bilateral hyperaldosteronism. Considerable progress has been made in defining the role for somatic gene mutations in aldosterone-producing adenomas (APA) as the primary cause of unilateral PA. This includes the use of next-generation sequencing (NGS) to define recurrent somatic mutations in APA that disrupt calcium signaling, increase aldosterone synthase (CYP11B2) expression, and aldosterone production. The use of CYP11B2 immunohistochemistry on adrenal glands from normal subjects, patients with unilateral and bilateral PA has allowed the identification of CYP11B2-positive cell foci, termed aldosterone-producing cell clusters (APCC). APCC lie beneath the adrenal capsule and like APA, many APCC harbor somatic gene mutations known to increase aldosterone production. These findings suggest that APCC may play a role in pathologic progression of PA. Herein, we provide an update on recent research directed at characterizing APCC and also discuss the unanswered questions related to the role of APCC in PA.

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.

Transgene-free remote magnetothermal regulation of adrenal hormones.

The field of bioelectronic medicines seeks to modulate electrical signaling within peripheral organs, providing temporally precise control of physiological functions. This is usually accomplished with implantable devices, which are often unsuitable for interfacing with soft and highly vascularized organs. Here, we demonstrate an alternative strategy for modulating peripheral organ function, which relies on the endogenous expression of a heat-sensitive cation channel, transient receptor potential vanilloid family member 1 (TRPV1), and heat dissipation by magnetic nanoparticles (MNPs) in remotely applied alternating magnetic fields. We use this approach to wirelessly control adrenal hormone secretion in genetically intact rats. TRPV1-dependent calcium influx into the cells of adrenal cortex and medulla is sufficient to drive rapid release of corticosterone and (nor)epinephrine. As altered levels of these hormones have been correlated with mental conditions such as posttraumatic stress disorder and major depression, our approach may facilitate the investigation of physiological and psychological impacts of stress.

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.

Chromaffin Cells of the Adrenal Medulla: Physiology, Pharmacology, and Disease.

Chromaffin cells (CCs) of the adrenal gland and the sympathetic nervous system produce the catecholamines (epinephrine and norepinephrine; EPI and NE) needed to coordinate the bodily "fight-or-flight" response to fear, stress, exercise, or conflict. EPI and NE release from CCs is regulated both neurogenically by splanchnic nerve fibers and nonneurogenically by hormones (histamine, corticosteroids, angiotensin, and others) and paracrine messengers [EPI, NE, adenosine triphosphate, opioids, γ-aminobutyric acid (GABA), etc.]. The "stimulus-secretion" coupling of CCs is a Ca2+ -dependent process regulated by Ca2+ entry through voltage-gated Ca2+ channels, Ca2+ pumps, and exchangers and intracellular organelles (RE and mitochondria) and diffusible buffers that provide both Ca2+ -homeostasis and Ca2+ -signaling that ultimately trigger exocytosis. CCs also express Na+ and K+ channels and ionotropic (nAChR and GABAA ) and metabotropic receptors (mACh, PACAP, ß-AR, 5-HT, histamine, angiotensin, and others) that make CCs excitable and responsive to autocrine and paracrine stimuli. To maintain high rates of E/NE secretion during stressful conditions, CCs possess a large number of secretory chromaffin granules (CGs) and members of the soluble NSF-attachment receptor complex protein family that allow docking, fusion, and exocytosis of CGs at the cell membrane, and their recycling. This article attempts to provide an updated account of well-established features of the molecular processes regulating CC function, and a survey of the as-yet-unsolved but important questions relating to CC function and dysfunction that have been the subject of intense research over the past 15 years. Examples of CCs as a model system to understand the molecular mechanisms associated with neurodegenerative diseases are also provided. Published 2019. Compr Physiol 9:1443-1502, 2019.