scholarly journals Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells.

1994 ◽  
Vol 127 (5) ◽  
pp. 1419-1433 ◽  
Author(s):  
Y Liu ◽  
E S Schweitzer ◽  
M J Nirenberg ◽  
V M Pickel ◽  
C J Evans ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Gradient Centrifugation ◽  
Secretory Vesicle ◽  
Dense Core ◽  
Gamma Aminobutyric Acid ◽  
Dense Core Vesicles ◽  
Protein Marker ◽  
Preferential Localization ◽  
Large Dense Core Vesicles ◽  
Pheochromocytoma Pc12

Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH-terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs.

Biosynthesis of large dense-core vesicles in PC12 cells: effects of depolarization and second messengers on the mRNA levels of their constituents

1995 ◽  
Vol 31 (1-2) ◽  
pp. 131-140 ◽  
Author(s):  
Claudia Tschernitz ◽  
Andrea Laslop ◽  
Christine Eiter ◽  
Stephan Kroesen ◽  
Hans Winkler
Keyword(s):  
Pc12 Cells ◽  
Second Messengers ◽  
Dense Core ◽  
Mrna Levels ◽  
Dense Core Vesicles ◽  
Large Dense Core Vesicles

The GTPase Rab3a is associated with large dense core vesicles in bovine chromaffin cells and rat PC12 cells

1995 ◽  
Vol 108 (4) ◽  
pp. 1639-1649 ◽  
Author(s):  
F. Darchen ◽  
J. Senyshyn ◽  
W.H. Brondyk ◽  
D.J. Taatjes ◽  
R.W. Holz ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Pc12 Cells ◽  
Immunoelectron Microscopy ◽  
Chromaffin Cells ◽  
Secretory Granules ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Large Dense Core Vesicles ◽  
Bovine Chromaffin Cells ◽  
Specific Association

Small GTPases of the rab family control intracellular vesicle traffic in eukaryotic cells. Although the molecular mechanisms underlying the activity of the Rab proteins have not been elucidated yet, it is known that the function of these proteins is dependent on their precise subcellular localization. It has been suggested that Rab3a, which is mainly expressed in neural and endocrine cells, might regulate exocytosis. Recently, direct experimental evidence supporting this hypothesis has been obtained. Consistent with such a role for Rab3a in regulated exocytosis was the previously reported specific association of Rab3a with synaptic vesicles and with secretory granules in adrenal chromaffin cells. Since the latter result, based on subcellular fractionation, has been controversial, we have re-investigated the subcellular localization of this GTP-binding protein by using a combination of morphological techniques. Bovine chromaffin cells were labelled with an affinity-purified polyclonal anti-Rab3a antibody and analyzed by confocal microcopy. Rab3a was found to colocalize partially with dopamine beta-hydroxylase, a chromaffin granule marker. In agreement with this observation, immunoelectron microscopy revealed a specific staining of chromaffin granules. In addition to large dense core vesicles, some small vesicles were labelled. To eliminate the possibility that the staining was due to a Rab3a-related protein, we investigated by immunoelectron microscopy the localization of an epitope-tagged Rab3a expressed in rat PC12 cells. Secretory granules were specifically labelled, whereas clear microvesicles were not. These results provide further evidence supporting a specific association of the GTPase Rab3a with large dense core secretory vesicles.

scholarly journals Vacuolar H+-ATPase subunits Voa1 and Voa2 cooperatively regulate secretory vesicle acidification, transmitter uptake, and storage

2011 ◽  
Vol 22 (18) ◽  
pp. 3394-3409 ◽  
Author(s):  
Ner Mu Nar Saw ◽  
Soo-Young Ann Kang ◽  
Leon Parsaud ◽  
Gayoung Anna Han ◽  
Tiandan Jiang ◽  
...  
Keyword(s):  
Pc12 Cells ◽  
Secretory Vesicle ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Functional Roles ◽  
Early Endosomes ◽  
Atpase Subunits ◽  
Peptide Secretion ◽  
Transmitter Uptake ◽  
And Storage

The Vo sector of the vacuolar H+-ATPase is a multisubunit complex that forms a proteolipid pore. Among the four isoforms (a1–a4) of subunit Voa, the isoform(s) critical for secretory vesicle acidification have yet to be identified. An independent function of Voa1 in exocytosis has been suggested. Here we investigate the function of Voa isoforms in secretory vesicle acidification and exocytosis by using neurosecretory PC12 cells. Fluorescence-tagged and endogenous Voa1 are primarily localized on secretory vesicles, whereas fluorescence-tagged Voa2 and Voa3 are enriched on the Golgi and early endosomes, respectively. To elucidate the functional roles of Voa1 and Voa2, we engineered PC12 cells in which Voa1, Voa2, or both are stably down-regulated. Our results reveal significant reductions in the acidification and transmitter uptake/storage of dense-core vesicles by knockdown of Voa1 and more dramatically of Voa1/Voa2 but not of Voa2. Overexpressing knockdown-resistant Voa1 suppresses the acidification defect caused by the Voa1/Voa2 knockdown. Unexpectedly, Ca2+-dependent peptide secretion is largely unaffected in Voa1 or Voa1/Voa2 knockdown cells. Our data demonstrate that Voa1 and Voa2 cooperatively regulate the acidification and transmitter uptake/storage of dense-core vesicles, whereas they might not be as critical for exocytosis as recently proposed.

EGFP-Tagged Vasopressin Precursor Protein Sorting Into Large Dense Core Vesicles and Secretion From PC12 Cells

2005 ◽  
Vol 25 (3-4) ◽  
pp. 581-605 ◽  
Author(s):  
Bing-Jun Zhang ◽  
Mitsuo Yamashita ◽  
Ray Fields ◽  
Kiyoshi Kusano ◽  
Harold Gainer
Keyword(s):  
Pc12 Cells ◽  
Protein Sorting ◽  
Precursor Protein ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Large Dense Core Vesicles ◽  
Vasopressin Precursor

scholarly journals Role of Secretory Carrier Membrane Protein SCAMP2 in Granule Exocytosis

2002 ◽  
Vol 13 (12) ◽  
pp. 4266-4278 ◽  
Author(s):  
Lixia Liu ◽  
Zhenheng Guo ◽  
Quyen Tieu ◽  
Anna Castle ◽  
David Castle
Keyword(s):  
Pc12 Cells ◽  
Plasma Membranes ◽  
Point Mutations ◽  
Specific Effect ◽  
Dense Core ◽  
Dense Core Vesicles ◽  
Granule Exocytosis ◽  
Structural Selectivity ◽  
Large Dense Core Vesicles ◽  
Dependent Inhibition

In secretory carrier membrane proteins (SCAMPs), the most conserved structural segment is between transmembrane spans 2 and 3, facing the cytosol. A synthetic peptide, CWYRPIYKAFR (E peptide), from this segment of SCAMP2 potently inhibits exocytosis in permeabilized neuroendocrine (PC12) cells. E peptide blocked discharge of35S-labeled secretogranin with the same structural selectivity and potency as observed for hexosaminidase secretion in mast cells. SCAMPs 1 and 2 are concentrated primarily on intracellular membranes in PC12 cells. Both, however, are found on plasma membranes, but neither is present on large dense-core vesicles. Yet, large dense-core vesicles marked by secretogranin attach to plasma membranes at foci containing SCAMP2 along with syntaxin1 and complexin at putative cell-surface docking/fusion sites. Regulated overexpression of SCAMP2 with point mutations in its E peptide but not of normal SCAMP2 caused dose-dependent inhibition of depolarization-induced secretion. The SCAMP2 mutants also inhibited secretion stimulated by elevated calcium. Inhibition was largely overcome by adding lysophosphatidylcholine to the medium at concentrations that do not otherwise affect secretion. Although overexpression of normal or mutant SCAMP2 slightly inhibits endocytosis, this effect does not appear to be related to the specific effect of the mutant SCAMP on stimulated exocytosis. Thus, SCAMP2 not only colocalizes with fusion sites but also appears to have an essential function in granule exocytosis through actions mediated by its E peptide–containing domain.

Sign in / Sign up

Export Citation Format

Share Document