Chromogranins (Cgs) are the major soluble proteins of dense-core secretory vesicles. Chromaffin cells fromChganull mice [chromogranin A knock-out (CgA-KO)] exhibited ∼30% reduction in the content and in the release of catecholamines compared with wild type. This was because of a lower secretion per single exocytotic event, rather than to a lower frequency of exocytotic events. Cell incubation withl-DOPA produced an increase in the vesicular amine content of wild-type, but not CgA-KO vesicles. In contrast, intracellular electrochemistry showed thatl-DOPA produced a significantly larger increase in cytosolic amines in CgA-KO cells than in the wild type. These data indicate that the mechanisms for vesicular accumulation in CgA-KO cells were fully saturated. Patch-amperometry recordings showed a delayed initiation of the amperometric signal after vesicle fusion, whereas no changes were observed in vesicle size or fusion pore kinetics despite the smaller amine content. We conclude that intravesicular proteins are highly efficient systems directly implicated in transmitter accumulation and in the control of neurosecretion.

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.

Several drugs produce rapid changes in the kinetics of exocytosis of catecholamines, as measured at the single event level with amperometry. This study is intended to unveil whether the mechanism(s) responsible for these effects involve changes in the intravesicular pH. Cell incubation with bafilomycin A1, a blocker of the vesicular proton pump, caused both a deceleration in the kinetics of exocytosis and a reduction in the catecholamine content of vesicle. These effects were also observed upon reduction of proton gradient by nigericin or NH₄Cl. pH measurements using fluorescent probes (acridine orange, quinacrine or enhanced green fluorescent protein–synaptobrevin) showed a strong correlation between vesicular pH and the kinetics of exocytosis. Hence, all maneuvers tested that decelerated exocytosis also alkalinized secretory vesicles and vice versa. On the other hand, calcium entry caused a transient acidification of granules. We therefore propose that the regulation of vesicular pH is, at least partially, a necessary step in the modulation of the kinetics of exocytosis and quantal size operated by some cell signals.

The effects of nitric oxide (NO) on the late phase of exocytosis have been studied, by amperometry, on Ba²⁺-stimulated chromaffin cells. Acute incubation with NO or NO donors (sodium nitroprusside, spermine-NO,S-nitrosoglutathione) produced a drastic slowdown of the granule emptying. Conversely, cell treatment with N^ω-nitro-l-arginine methyl ester (a NO synthase inhibitor) or with NO scavengers (methylene blue, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium) accelerated the extrusion of catecholamines from chromaffin granules, suggesting the presence of a NO modulatory tone. The incubation with phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine or zaprinast) or with the cell-permeant cGMP analog 8-bromo-cGMP, mimicked the effects of NO, suggesting the involvement of the guanylate cyclase cascade. NO effects were not related to changes in intracellular Ba²⁺. NO did not modify the duration of feet. Effects were evident even on pre-fusioned granules, observed under hypertonic conditions, suggesting that the fusion pore is not the target for NO, which probably acts by modifying the affinity of catecholamines for the intragranular matrix. NO could modify the synaptic transmitter efficacy through a novel mechanism, which involves the regulation of the emptying of secretory vesicles.

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