Modification of annexin II expression in PC12 cell lines does not affect Ca(2+)-dependent exocytosis.

The Ca2+/phospholipid/cytoskeletal-binding protein annexin II has been proposed to play an important role in Ca(2+)-dependent exocytosis; however, the evidence for this role is inconclusive. More direct evidence obtained by manipulating annexin II levels in cells is still required. We have attempted to do this by generating stably transfected PC12 cell lines expressing proteins which elevate or lower functional annexin II levels and using these cell lines to investigate Ca(2+)-dependent exocytosis. Three cell lines were generated: one expressing an annexin II mutant which aggregates annexin II in at least a proportion of the cells, thereby removing functional protein from the cell; a mixed clonal cell line constitutively overexpressing human annexin II; and a clonal cell line capable of over-expressing annexin II in the presence of sodium butyrate. After digitonin permeabilization, Ca(2+)-dependent dopamine release from these cell lines was compared with that from control nontransfected cells, and, in addition, release was compared in induced to uninduced cells. There were no significant differences in Ca(2+)-dependent exocytosis between any of the transfected cell lines before or after induction and the control cells. In addition, nontransfected PC12 cells treated with nerve growth factor, which elevates annexin II levels severalfold, failed to increase Ca(2+)-dependent exocytosis after digitonin permeabilization, compared with control cells. We conclude that annexin II is not an important regulator of Ca(2+)-dependent exocytosis in PC12 cells.

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Pituitary adenylate cyclase-activating polypeptide releases 7B2, adrenocorticotrophin, growth hormone and prolactin from the mouse and rat clonal pituitary cell lines AtT-20 and GH3

Journal of Endocrinology ◽

10.1677/joe.0.1320107 ◽

1992 ◽

Vol 132 (1) ◽

pp. 107-113 ◽

Cited By ~ 50

Author(s):

R. Propato-Mussafiri ◽

S. M. Kanse ◽

M. A. Ghatei ◽

S. R. Bloom

Keyword(s):

Adenylate Cyclase ◽

Cell Line ◽

Cell Lines ◽

Proteolytic Processing ◽

Somatostatin Analogue ◽

Acth Secretion ◽

Clonal Cell Line ◽

Thyrotrophin Releasing Hormone ◽

Clonal Cell ◽

Pituitary Adenylate Cyclase

ABSTRACT Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from ovine hypothalami and so called because of its ability to stimulate pituitary adenylate cyclase activity. Alternative amidation and proteolytic processing of prepro-PACAP gives rise to two bioactive-amidated forms, PACAP-NH2(1–38) (PACAP-38) and PACAP-NH2(1–27) (PACAP-27). 7B2 is a polypeptide of 185 amino acids which is predominantly found in secretory granules and is widely distributed in rat and human tissues. We investigated the ability of the two forms of PACAP to stimulate GH, prolactin and 7B2 release by the rat pituitary clonal cell line GH3, and ACTH and 7B2 by the mouse pituitary clonal cell line AtT-20. PACAP-38 and PACAP-27 stimulated 7B2 and GH/prolactin or ACTH secretion with a similar efficacy over the 2-h incubation period from GH3 and AtT-20 cells respectively. 7B2 secretion was also stimulated by corticotrophin-releasing factor (CRF-41) and vasoactive intestinal polypeptide (VIP) in AtT-20 cells, and thyrotrophin-releasing hormone (TRH) and VIP in GH3 cells. Addition of PACAP to CRF-41 resulted in an additive effect on ACTH secretion and a synergistic effect on 7B2 secretion in AtT-20 cells. No synergism was observed when PACAP was added together with TRH, either on GH and prolactin secretion or on 7B2 release from GH3 cells. PACAP-mediated 7B2 secretion from both cell lines and PACAP-stimulated ACTH release from AtT-20 cells were reduced by 5 mg octapeptide synthetic somatostatin analogue/l (5 mg SMS 201-995/1). Journal of Endocrinology (1992) 132, 107–113

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Binding of a tritiated thyrotropin-releasing factor to a prolactin secreting clonal cell line (GH3)

Experimental Cell Research ◽

10.1016/0014-4827(73)90242-5 ◽

1973 ◽

Vol 82 (1) ◽

pp. 39-46 ◽

Cited By ~ 51

Author(s):

Danielle Gourdji ◽

A. Tixier-Vidal ◽

Annie Morin ◽

P. Pradelles ◽

J.-L. Morgat ◽

...

Keyword(s):

Cell Line ◽

Clonal Cell Line ◽

Clonal Cell

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Perifusion of a Clonal Cell Line of Simian Virus 40-Transformed Beta Cells: Insulin Secretory Dynamics in Response to Glucose, 3-Isobutyl-1-Methylxanthine, and Potassium

Diabetes ◽

10.2337/diab.34.2.115 ◽

1985 ◽

Vol 34 (2) ◽

pp. 115-120 ◽

Cited By ~ 26

Author(s):

R. S. Hill ◽

A. E. Boyd

Keyword(s):

Cell Line ◽

Beta Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

Clonal Cell Line ◽

Clonal Cell

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Growth of a clonal cell line of Helicoverpa zea (Lepidoptera: Noctuidae) in suspension culture and replication of its homologous baculovirus HzSNPV.

Applied Entomology and Zoology ◽

10.1303/aez.2001.349 ◽

2001 ◽

Vol 36 (3) ◽

pp. 349-352 ◽

Cited By ~ 6

Author(s):

Arthur H. McIntosh ◽

James J. Grasela ◽

Cynthia L. Goodman ◽

Carlo M. Ignoffo

Keyword(s):

Suspension Culture ◽

Cell Line ◽

Helicoverpa Zea ◽

Clonal Cell Line ◽

Clonal Cell ◽

Lepidoptera Noctuidae

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Synaptotagmin VII Is Targeted to Dense-core Vesicles and Regulates Their Ca2+-dependent Exocytosis in PC12 Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m409241200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 52677-52684 ◽

Cited By ~ 58

Author(s):

Mitsunori Fukuda ◽

Eiko Kanno ◽

Megumi Satoh ◽

Chika Saegusa ◽

Akitsugu Yamamoto

Keyword(s):

Plasma Membrane ◽

Neuropeptide Y ◽

Cell Line ◽

Pc12 Cells ◽

Pc12 Cell ◽

Small Interfering Rna ◽

Dense Core ◽

Dense Core Vesicles ◽

Pc12 Cell Line ◽

Interfering Rna

It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membraneversussecretory granules). In this study we established a PC12 cell line “stably expressing” the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as thetrans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+-dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (∼60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (∼85–90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expressionversusstable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+sensor for exocytosis in endocrine cells.

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