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.

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.exocytosis | purines | quantum size | secretory vesicles | VNUT V irtually most, and possibly all, types of secretory vesicles found in cells contain ATP, which often accumulates at high concentrations and, commonly, in conjunction with different types of neurotransmitters. However, the reason for this widespread distribution of ATP remains a mystery. Although ATP is present in all animal species, including primitive life forms like Giardia lamblia that lack Golgi complexes and mitochondria, the detection of ATP in the secretory vesicles of sympathetic neurons was considered to be the first example of cotransmission (1). However, given the ubiquitous accumulation of ATP in secretory vesicles, it might instead be considered that it is the other neurotransmitters that coincide with ATP, rather than the other way around (2). Indeed, perhaps ATP should be considered as the first molecule used as a transmitter in primitive forms of life.Astonishingly high concentrations of releasable species are stored inside secretory vesicles, far exceeding those in the cytosol (3, 4). For example, the catecholamine content of adrenal secretory granules (SGs), a type of large dense core secretory vesicles also known as chromaffin granules, was 0.8-1 M when measured directly in adrenal chromaffin cells using patch amperometry (5, 6). In addition, SGs from chromaffin cells contain ATP at ∼150 mM (7), calcium ∼40 mM (8), about 2 mM of granins, ascorbate, peptides, and other nucleotides, all in an acidic pH ∼5.5 environment.ATP possesses intrinsic chemical characteristics that make it relevant to the accumulation of soluble substances in secretory vesicles. The formation of weak complexes between monoamines and ATP, the two main soluble compounds in chromaffin granules, has been demonstrated in vitro by NMR (9), ultracentrifugation (10), infrared spectroscopy, and calorime...

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.

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.

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, patchamperometry 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.