Pool size estimations for dense-core vesicles in mammalian CNS neurons.

Neuropeptides are essential signaling molecules transported and secreted by dense-core vesicles (DCVs), but the number of DCVs available for secretion, their subcellular distribution, and release probability are unknown. Here, we quantified DCV pool sizes in three types of mammalian CNS neurons in vitro and in vivo Super-resolution and electron microscopy reveal a total pool of 1,400-18,000 DCVs, correlating with neurite length. Excitatory hippocampal and inhibitory striatal neurons in vitro have a similar DCV density, and thalamo-cortical axons in vivo have a slightly higher density. Synapses contain on average two to three DCVs, at the periphery of synaptic vesicle clusters. DCVs distribute equally in axons and dendrites, but the vast majority (80%) of DCV fusion events occur at axons. The release probability of DCVs is 1-6%, depending on the stimulation. Thus, mammalian CNS neurons contain a large pool of DCVs of which only a small fraction can fuse, preferentially at axons.

Adrenergic chromaffin cells are adrenergic even in the absence of epinephrine.

Adrenal chromaffin cells release epinephrine (EPI) and norepinephrine (NE) into the bloodstream as part of the homeostatic response to situations like stress. Here we utilized EPI-deficient mice generated by knocking out (KO) the phenylethanolamine N-methyltransferase (Pnmt) gene. These Pnmt-KO mice were bred to homozygosis but displayed no major phenotype. The lack of EPI was partially compensated by an increase in NE, suggesting that EPI storage was optimized in adrenergic cells. Electron microscopy showed that despite the lack of EPI, chromaffin granules retain their shape and general appearance. This indicate that granules from adrenergic or noradrenergic cells preserve their characteristics even though they contain only NE. Acute insulin injection largely reduced the EPI content in wild-type animals, with a minimal reduction in NE, whereas there was only a partial reduction in NE content in Pnmt-KO mice. The analysis of exocytosis by amperometry revealed a reduction in the quantum size (-30%) and Imax (-21%) of granules in KO cells relative to the wild-type granules, indicating a lower affinity of NE for the granule matrix of adrenergic cells. As amperometry cannot distinguish between adrenergic or noradrenergic cells, it would suggest even a larger reduction in the affinity for the matrix. Therefore, our results demonstrate that adrenergic cells retain their structural characteristics despite the almost complete absence of EPI. Furthermore, the chromaffin granule matrix from adrenergic cells is optimized to accumulate EPI, with NE being a poor substitute. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

ATP: The crucial component of secretory vesicles.

The colligative properties of ATP and catecholamines demonstrated in vitro are thought to be responsible for the extraordinary accumulation of solutes inside chromaffin cell secretory vesicles, although this has yet to be demonstrated in living cells. Because functional cells cannot be deprived of ATP, we have knocked down the expression of the vesicular nucleotide carrier, the VNUT, to show that a reduction in vesicular ATP is accompanied by a drastic fall in the quantal release of catecholamines. This phenomenon is particularly evident in newly synthesized vesicles, which we show are the first to be released. Surprisingly, we find that inhibiting VNUT expression also reduces the frequency of exocytosis, whereas the overexpression of VNUT drastically increases the quantal size of exocytotic events. To our knowledge, our data provide the first demonstration that ATP, in addition to serving as an energy source and purinergic transmitter, is an essential element in the concentration of catecholamines in secretory vesicles. In this way, cells can use ATP to accumulate neurotransmitters and other secreted substances at high concentrations, supporting quantal transmission.

How intravesicular composition affects exocytosis.

Large dense core vesicles and chromaffin granules accumulate solutes at large concentrations (for instance, catecholamines, 0.5-1 M; ATP, 120-300 mM; or Ca2+, 40 mM (12)). Solutes seem to aggregate to a condensed protein matrix, which is mainly composed of chromogranins, to elude osmotic lysis. This association is also responsible for the delayed release of catecholamines during exocytosis. Here, we compile experimental evidence, obtained since the inception of single-cell amperometry, demonstrating how the alteration of intravesicular composition promotes changes in the quantum characteristics of exocytosis. As chromaffin cells are large and their vesicles contain a high concentration of electrochemically detectable species, most experimental data comes from this cell model.

Distinct patterns of exocytosis elicited by Ca2+, Sr2+ and Ba2+ in bovine chromaffin cells.

Three divalent cations can elicit secretory responses in most neuroendocrine cells, including chromaffin cells. The extent to which secretion is elicited by the cations in intact depolarized cells was Ba2+ > Sr2+ ≥ Ca2+, contrasting with that elicited by these cations in permeabilized cells (Ca2+ > Sr2+ > Ba2+). Current-clamp recordings show that extracellular Sr2+ and Ba2+ cause membrane depolarization and action potentials, which are not blocked by Cd2+ but that can be mimicked by tetra-ethyl-ammonium. When applied intracellularly, only Ba2+ provokes action potentials. Voltage-clamp monitoring of Ca2+-activated K+ channels (KCa) shows that Ba2+ reduces outward currents, which were enhanced by Sr2+. Extracellular Ba2+ increases cytosolic Ca2+ concentrations in Fura-2-loaded intact cells, and it induces long-lasting catecholamine release. Conversely, amperometric recordings of permeabilized cells show that Ca2+ promotes the longest lasting secretion, as Ba2+ only provokes secretion while it is present and Sr2+ induces intermediate-lasting secretion. Intracellular Ba2+ dialysis provokes exocytosis at concentrations 100-fold higher than those of Ca2+, whereas Sr2+ exhibits an intermediate sensitivity. These results are compatible with the following sequence of events: Ba2+ blocks KCa channels from both the outside and inside of the cell, causing membrane depolarization that, in turn, opens voltage-sensitive Ca2+ channels and favors the entry of Ca2+ and Ba2+. Although Ca2+ is less permeable through its own channels, it is more efficient in triggering exocytosis. Strontium possesses both an intermediate permeability and an intermediate ability to induce secretion.

Dense-core vesicle biogenesis and exocytosis in neurons lacking chromogranins A and B.

Chromogranin A and B (Cgs) are considered to be master regulators of cargo sorting for the regulated secretory pathway (RSP) and dense-core vesicle (DCV) biogenesis. To test this, we analyzed the release of neuropeptide Y (NPY)-pHluorin, a live RSP reporter, and the distribution, number, and appearance of DCVs, in mouse hippocampal neurons lacking expression of CHGA and CHGB genes. qRT-PCR analysis showed that expression of other granin family members was not significantly altered in CgA/B-/- neurons. As synaptic maturation of developing neurons depends on secretion of trophic factors in the RSP, we first analyzed neuronal development in standardized neuronal cultures. Surprisingly, dendritic and axonal length, arborization, synapse density, and synaptic vesicle accumulation in synapses were all normal in CgA/B-/- neurons. Moreover, the number of DCVs outside the soma, stained with endogenous marker Secretogranin II, the number of NPY-pHluorin puncta, and the total amount of reporter in secretory compartments, as indicated by pH-sensitive NPY-pHluorin fluorescence, were all normal in CgA/B-/- neurons. Electron microscopy revealed that synapses contained a normal number of DCVs, with a normal diameter, in CgA/B-/- neurons. In contrast, CgA/B-/- chromaffin cells contained fewer and smaller secretory vesicles with a smaller core size, as previously reported. Finally, live-cell imaging at single vesicle resolution revealed a normal number of fusion events upon bursts of action potentials in CgA/B-/- neurons. These events had normal kinetics and onset relative to the start of stimulation. Taken together, these data indicate that the two chromogranins are dispensable for cargo sorting in the RSP and DCV biogenesis in mouse hippocampal neurons.

The interaction between chromogranin A and catecholamines governs exocytosis.

Chromogranins (Cgs) are acidic proteins that have been described in the large, dense core vesicles (LDCVs) of adrenal chromaffin cells and that have been shown to promote LDCV formation, even in nonsecretory cells. Catecholamines (CAs) are adsorbed by Cgs in vitro, and the absence of Cgs modifies the storage and exocytosis of CAs in chromaffin cells. In this study, we set out to assess the role of CgA in the accumulation and exocytosis of CAs in cells when the levels of CgA and CA are manipulated. We overexpressed CgA in nonsecretory HEK293 cells and in secretory PC12 cells, to study the formation, movement, and exocytosis of newly formed granules by evanescent wave microscopy. We analyzed the association of Cgs/CA by HPLC and amperometry and their role in the accumulation and exocytosis of amines, both under resting conditions and after l-DOPA overloading. To our knowledge, this is the first demonstration that CgA expression in a nonsecretory cell line facilitates the storage and exocytosis of CA. In addition, CgA overexpression causes a doubling of the accumulation of CA, although it slows down exocytosis in PC12 cells. We propose a model to explain how the CgA/CA complex governs the accumulation and exocytosis of secreted amines.

Chromogranins A and B are key proteins in amine accumulation, but the catecholamine secretory pathway is conserved without them.

Chromogranins are the main soluble proteins in the large dense core secretory vesicles (LDCVs) found in aminergic neurons and chromaffin cells. We recently demonstrated that chromogranins A and B each regulate the concentration of adrenaline in chromaffin granules and its exocytosis. Here we have further studied the role played by these proteins by generating mice lacking both chromogranins. Surprisingly, these animals are both viable and fertile. Although chromogranins are thought to be essential for their biogenesis, LDCVs were evident in these mice. These vesicles do have a somewhat atypical appearance and larger size. Despite their increased size, single-cell amperometry recordings from chromaffin cells showed that the amine content in these vesicles is reduced by half. These data demonstrate that although chromogranins regulate the amine concentration in LDCVs, they are not completely essential, and other proteins unrelated to neurosecretion, such as fibrinogen, might compensate for their loss to ensure that vesicles are generated and the secretory pathway conserved.

Quantal Release Analysis of Electrochemically Active Molecules Using Single-Cell Amperometry.

Single-cell amperometry is a powerful technique that permits the detection of electrochemically active transmitters, such as catecholamines, histamine, or serotonin, released by exocytosis from secretory cells.Amperometry has two main characteristics that make it ideal for the study of exocytosis at the single-cell level with single-vesicle resolution quantal release. (i) It is noninvasive. The carbon fiber microelectrode can be carefully positioned on plasma membrane of a single cell, allowing the detection of the oxidation current of the secreted molecules. (ii) High temporal resolution and sensitivity. Exocytosis can be monitored with a real-time resolution that allows the determination of the kinetics release with an attomol detection sensitivity, which ensures an accurate calculation of the amount of transmitter released.Here, we compile some recommendations and advices to perform amperometry quantal analysis.

Nodular (keloidal) scleroderma.

Keloidal or nodular scleroderma (NS) is a variant of localized scleroderma (LS) frequently seen in patients with limited or diffuse systemic sclerosis (SSc). It presents as raised, firm plaques or nodules with extensive dermal fibrosis and hyalinized collagen bundles. We present a patient with SSc who presented with this rare entity.

Chromogranins as regulators of exocytosis.

Chromogranins (Cgs) constitute the main protein component in the vesicular matrix of large dense core vesicles (LDCV). These acidic proteins have been implicated in several physiological processes such as vesicle sorting, the generation of bioactive peptides and the accumulation of soluble species inside LDCV. This latter feature of Cgs accounts for the ability of vesicles to concentrate catecholamines and Ca(2+). Indeed, the low affinity and high capacity of Cgs to bind solutes at the low pH of the LDCV lumen seems to be behind the delay in the neurotransmitter exit towards the extracellular milieu after vesicle fusion. The availability of new mouse strains lacking Cgs in combination with the arrival of several techniques for the direct monitoring of exocytosis (like amperometry, patch-amperometry and intracellular electrochemistry), have helped advance our understanding of how these granins concentrate catecholamines and Ca(2+) in LDCV, and how they influence the kinetics of exocytosis. In this review, we will discuss the roles of Cgs A and B in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from adrenal chromaffin cells.

Chromogranins A and B as regulators of vesicle cargo and exocytosis.

Chromogranins (Cgs) are acidic proteins that have been implicated in several physiological processes such as vesicle sorting, the production of bioactive peptides and the accumulation of soluble species inside large dense core vesicles (LDCV). They constitute the main protein component in the vesicular matrix of LDCV. This latter characteristic of Cgs accounts for the ability of vesicles to concentrate catecholamines and Ca(2+). It is likely that Cgs are behind the delay in the neurotransmitter exit towards the extracellular milieu after vesicle fusion, due to their low affinity and high capacity to bind solutes present inside LDCV. The recent availability of mouse strains lacking Cgs, combined with the arrival of several techniques for the direct monitoring of exocytosis, have helped to expand our knowledge about the mechanisms used by granins to concentrate catecholamines and Ca(2+) in LDCV, and how they affect the kinetics of exocytosis. We will discuss the roles of Cgs A and B in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from adrenal chromaffin cells.

[Vulvar syringoma: a report of two cases]. / Siringomas vulvares: informe de dos casos.

Syringoma is a benign neoplasm of eccrine origin. Lesions often arise around the eyes and on the upper cheeks of middle-aged women. Syringomas involving the genitalia are rare, they present as multiple, bilateral, skin-colored papules over the labia majora. The lesions should therefore be considered in the differential diagnosis of multiple papular lesions of the vulva. We report two females of 37 and 62 years of age, both observed in a dermatological service.

Mice lacking chromogranins exhibit increased aggressive and depression-like behaviour.

Chromogranins are acidic proteins; both chromogranins A and B constitute the main protein component in the vesicular matrix of large dense core vesicles. Chromogranins are a natural source of peptides with different physiological activities that have been associated with vascular and neurological diseases. We have used three different genetic mutant models of mice lacking chromogranin A, chromogranin B and both all on the same C57BL/6J background, to characterize the physiological roles of these proteins using metabolic, cardiovascular and behavioural tests. In mice from 3 to 18 months of age, the lack of any chromogranin promoted age-dependent hypersensitivity to insulin, while the lack of both chromogranins provoked progressive lack of response to stress, as restriction did not promote tachycardia in old mice. Moreover, the lack of chromogranin B produced a depressive-like and aggressive phenotype, while the lack either or both chromogranins increased barbering behaviour. In addition, we observed no effects on light-dark box or RotaRod tests. Mice lacking chromogranin B exhibited lower exploratory activity. Based on this extensive phenotyping with more than 2800 mice, these findings support roles of chromogranins, or the peptides derived from them, in the control of aggressive behaviour along with changes in their metabolic profile beyond their previously described activities in the secretory pathway.

The role of chromogranins in the secretory pathway.

Chromogranins (Cgs) are acidic proteins implicated in several physiological processes, including the biogenesis and sorting of secretory vesicles, the generation of bioactive peptides, and the accumulation of soluble species inside large dense core vesicles (LDCV). Indeed, Cgs are the main protein component of the vesicular matrix in LDCV, and they are involved in the concentration of soluble species like neurotransmitters and calcium. Experiments using electrochemical techniques such amperometry, patch amperometry, and intracellular electrochemistry have clarified the functional roles of Cgs in the accumulation and release of catecholamines. We have focused this review at a single event of exocytosis of chromaffin cells from three mouse strains lacking Cgs. Accordingly, in this brief review, we will focus on the role of Cgs in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from studies on adrenal chromaffin cells.

Granins and catecholamines: functional interaction in chromaffin cells and adipose tissue.

Catecholamines (CAs) and granin peptides are costored in dense-core vesicles within the chromaffin cells of the adrenal medulla and in other endocrine organs and neurons. Granins play a major functional and structural role in chromaffin cells but are ubiquitous proteins, which are present also in secretory cells of the nervous, endocrine, and immune systems, where they regulate a number of cellular functions. Furthermore, recent studies also demonstrate that granin-derived peptides can functionally interact with CA to modulate key physiological functions such as lipolysis and blood pressure. In this chapter, we will provide a brief update on the interaction between CA and granins at the cellular and organ levels. We will first discuss recent data on the regulation of exocytosis of CA and peptides from the chromaffin cells by the sympathetic nervous system with a specific reference to the prominent role played by splanchnic nerve-derived pituitary adenylate cyclase-activating peptide (PACAP). Secondly, we will discuss the role of granins in the storage and regulation of exocytosis in large dense-core vesicles. Finally, we will provide an up-to-date review of the roles played by two granin-derived peptides, the chromogranin A-derived peptide catestatin and the VGF-derived peptide TLQP-21, on lipolysis and obesity. In conclusion, the knowledge gathered from recent findings on the role played by proteins/peptides in the sympathetic/target cell synapses, discussed in this chapter, would contribute to and provide novel mechanistic support for an increased appreciation of the physiological role of CA in human pathophysiology.

Preparation and culture of adrenal chromaffin cells.

Cultured chromaffin cells have been used for almost 40 years in the study of different cell functions using biochemical, electrophysiological, pharmacological, and toxicological approaches. Chromaffin cells are essentially secretory cells that are used to model sympathetic neurons or neuroendocrine cells. In this chapter, we describe the most common methods currently used to isolate and culture chromaffin cells from the animals used most commonly: cows, rats, and mice. We also provide some advice on the use of these cells in the laboratory.

Vesicular Ca(2+) mediates granule motion and exocytosis.

Secretory vesicles of chromaffin cells are acidic organelles that maintain an increasing pH gradient towards the cytosol (5.5 vs. 7.3) that is mediated by V-ATPase activity. This gradient is primarily responsible for the accumulation of large concentrations of amines and Ca(2+), although the mechanisms mediating Ca(2+) uptake and release from granules, and the physiological relevance of these processes, remain unclear. The presence of a vesicular matrix appears to create a bi-compartmentalised medium in which the major fractions of solutes, including catecholamines, nucleotides and Ca(2+), are strongly associated with vesicle proteins, particularly chromogranins. This association appears to be favoured at acidic pH values. It has been demonstrated that disrupting the pH gradient of secretory vesicles reduces their rate of exocytosis and promotes the leakage of vesicular amines and Ca(2+), dramatically increasing the movement of secretory vesicles and triggering exocytosis. In this short review, we will discuss the data available that highlights the importance of pH in regulating the association between chromogranins, vesicular amines and Ca(2+). We will also address the potential role of vesicular Ca(2+) in two major processes in secretory cells, vesicle movement and exocytosis.

The functional role of chromogranins in exocytosis.

Chromogranins A (CgA) and B (CgB) are the main soluble proteins of large dense-core secretory vesicles (LDCVs). Using CgA- and CgB-knockout (KO) mice, we found that the absence of chromogranins A and B induces significant changes in catecholamine (CA) accumulation and the kinetics of exocytosis. By crossing these two knockout strains, we generated a viable and fertile double CgA/B-KO mouse in which the catecholamine content in chromaffin LDCVs was halved, and the secretory response significantly reduced. Incubating cells with L-DOPA increased the vesicular CA content in wild-type (WT) but not in Cg-KO cells, which was not due to changes in amine transport, or in the synthesis or degradation of cytosolic amines. Electron microscopy revealed the presence of giant secretory vesicles exhibiting significant alterations, with little or no electrodense inner matrix. Proteomic analysis confirmed the absence of CgA and B, and revealed small changes in SgII in the LDCV-enriched fraction, as well as the overexpression of fibrinogen and other proteins. In summary, our findings indicate that the mechanisms responsible for vesicular accumulation of CA are saturated in Cgs-KO cells, in contrast to the ample capacity for further accumulation in WT cells. We conclude that Cgs contribute to a highly efficient system that directly mediates monoamine accumulation and exocytosis in LDCVs.