Natalia Domínguez

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.—Díaz‐Vera, J., Camacho, M., Machado, J. D., Domínguez, N., Montesinos, M. S., Hernández‐Fernaud, J. R., Luján, R., Borges, R. Chromogranins A and B are key proteins in amine accumulation, but the catecholamine secretory pathway is conserved without them. FASEB J. 26, 430–438 (2012). www.fasebj.org

Chromogranins as regulators of exocytosis

J. Neurochem. (2010) 114, 335–343.

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 Ca2+. 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 Ca2+ 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.

ATP: The crucial component of secretory vesicles

Significance ATP is highly concentrated in secretory vesicles. In vitro experiments suggest that the association of ATP with catecholamines reduces their osmotic forces, permitting the extraordinary accumulation of amines within chromaffin granules. However, this has yet to be proved in living cells. Because functional cells cannot be deprived of ATP, we manipulated the vesicular nucleotide carrier, demonstrating that the extent of vesicular ATP is closely linked to the quantum catecholamine size. This is particularly evident in newly synthesized vesicles, the first to be released. This is the in vivo demonstration that vesicular ATP is an essential factor in the accumulation of neurotransmitters, which may well be a wider mechanism supporting quantal transmission. 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.

Vesicular Ca2+ 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 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.—Dominguez, N., Estevez‐Herrera, J., Borges, R., Machado, J. D., The interaction between chromogranin A and catecholamines governs exocytosis. FASEB J. 28, 4657–4667 (2014). www.fasebj.org

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.

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.

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

Abstract 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.