Mouse Chromaffin Cells Have Two Populations of Dense Core Vesicles

Abstract

The quantal hypothesis states that neurotransmitter is released in discrete packages, quanta, thought to represent the neurotransmitter content of individual vesicles. If true, then vesicle size should influence quantal size. Although chromaffin cells are generally thought to have a single population of secretory vesicles, our electron microscopy analysis suggested two populations as the size distribution was best described as the sum of two Gaussians. The average volume difference was fivefold. To test whether this difference in volume affected quantal size, neurotransmitter release from permeabilized cells exposed to 100 microM Ca2+ was measured with amperometry. Quantal content was bimodally distributed with both large and small events; the distribution of vesicle sizes predicted by amperometry was extremely similar to those measured with electron microscopy. In addition, each population of events exhibited distinct release kinetics. These results suggest that chromaffin cells have two populations of dense core vesicles (DCV) with unique secretory properties and which may represent two distinct synthetic pathways for DCV biogenesis or alternatively they may represent different stages of biosynthesis.

Regulation of large dense-core vesicle volume and neurotransmitter content mediated by adaptor protein 3

“…Although it is possible that a fraction of the neurotransmitter content remained inside the vesicle, the strong correlation between vesicle size and quantal size suggests that the vesicle neurotransmitter content was completely emptied under the stimulation conditions used in this study. These findings are similar to previous studies performed in different cell types that have reported that vesicle size has a strong influence on quantal size (33,(37)(38)(39). Our studies are consistent with AP-3 participating in neurosecretory vesicle formation and secretion.…”

“…Previously, we found a strong link between amperometric event size and vesicle size in control C57BL͞6J mice under conditions similar to what we have used here (ref. 33 and unpublished observations). A morphological analysis of secretory vesicles in mocha and the appropriate control chromaffin cells was made to address the simplest scenario that vesicle size underlies the change in quantal size.…”

“…The neuronal-specific AP-3 subunits (referred to as AP-3B: ␦, ␤3〉, 3〉, and 3) and green fluorescent protein (GFP) were overexpressed in chromaffin cells. Secretion was initiated by exposing digitonin-permeabilized cells to a fixed, high concentration of Ca 2ϩ (100 M) (33). The amperometric traces show that a typical control cell (Fig.…”

“…Chromaffin cells were prepared similar to methods published in ref. 33. Cells were plated on glass coverslips coated with Matrigel (Discovery Labware, Bedford, MA) and maintained in a 37°C, 5% CO 2 incubator.…”

“…Under the intense stimulation conditions used here, recruitment for secretion is likely to bypass subtle differences in fusogenicity that may dominate under more physiological conditions; therefore, it is possible that the release of a broad spectrum of vesicles is induced with the intense stimulation used here. An entirely different explanation is that the cells have two separate populations of vesicles, as has been proposed for chromaffin cells (19,33) and PC12 cells (50). It is possible that AP-3 promotes the formation of smaller vesicles because overexpression of AP-3 increases the frequency of small events.…”