Neuropeptide release by efficient recruitment of diffusing cytoplasmic secretory vesicles

Abstract

Neuropeptides are slowly released from a limited pool of secretory vesicles. Despite decades of research, the composition of this pool has remained unknown. Endocrine cell studies support the hypothesis that a population of docked vesicles supports the first minutes of hormone release. However, it has been proposed that mobile cytoplasmic vesicles dominate the releasable neuropeptide pool. Here, to determine the cellular basis of the releasable pool, single green fluorescent protein-labeled secretory vesicles were visualized in neuronal growth cones with the use of an inducible construct or total internal reflection fluorescence microscopy. We report that vesicle movement follows the diffusion equation. Furthermore, rapidly moving secretory vesicles are used more efficiently than stationary vesicles near the plasma membrane to support stimulated release. Thus, randomly moving cytoplasmic vesicles participate in the first minutes of neuropeptide release. Importantly, the preferential recruitment of diffusing cytoplasmic secretory vesicles contributes to the characteristic slow kinetics and limited extent of sustained neuropeptide release.

Calcium-Dependent Exocytosis of Atrial Natriuretic Peptide from Astrocytes

“…These puncta represent ANPcontaining vesicles because immunocytochemical studies using an antibody against ANP and synaptobrevin II, a vesicleassociated protein, revealed colocalization of the green puncta with the signal from the ANP antibody and the synaptobrevin II antibody (data not shown). If ANP is released from astrocytes by regulated exocytosis, then the intensity of the green fluorescing probe should decrease (Han et al, 1999). Indeed, the stimulation of cells by the application of ionomycin to increase cytosolic [Ca 2ϩ ] resulted in an apparent decline in the number of green fluorescing puncta.…”

“…The release of ANP from a single cell was studied by transfecting cells with a construct to express ANP.emd, which was shown previously to be targeted to secretory vesicles (Han et al, 1999). Figure 1 shows that, in transfected rat astrocytes, green fluorescence appears in puncta throughout the cytoplasm.…”

“…To this end, confocal microscopy was used. To monitor the release of the neuropeptide, we transfected cells with a construct to express ANP.emd (Han et al, 1999). In our successfully transfected cells, fluorescence appeared as discrete puncta.…”

“…The plasmid encoding atrial natriuretic factor tagged with emerald green fluorescent protein, ANP.emd (a gift of Dr. Ed Levitan, University of Pittsburgh, Pittsburgh, PA) (Han et al, 1999), was introduced into the astrocytes by means of lipofection using the standard reference protocol of Invitrogen (Carlsbad, CA). DNA was mixed with 6 l of Plus Reagent (Invitrogen), diluted in 100 l of serum-free DMEM, and incubated for 15 min at room temperature (RT).…”

“…Astrocytes also possess several components of secretory apparatus, such as synaptobrevin II, cellubrevin, syntaxin (Parpura et al, 1995), and SNAP-23 (synaptosome-associated protein of 23 kDa) (Hepp et al, 1999); hence, ANP could be released via Ca 2ϩ -dependent exocytosis. To study the mechanism of ANP release from astrocytes, we transfected cells with a construct to express pro-ANP fused with the emerald green fluorescent protein (ANP.emd) (Han et al, 1999).…”