A primary culture of parotid acinar cells retaining capacity for agonists-induced amylase secretion and generation of new secretory granules.
Exocrine acinar cells, like parotid cells, have difficulty in maintaining their functions in cell lines or in primary cultures. For this reason, molecular studies on exocrine cell functions are unsatisfactory. To examine the mechanisms whereby the functions of parotid acinar cells are maintained, we attempted to establish a system for primary culture and transfection of exogenous genes. Acinar cells were dispersed from rat parotid glands by digestion with enzymes and were cultured in a medium containing rat serum. Most of the cultured cells had secretory granules that contained amylase, suggesting that they were derived from acinar cells, although they spread on the dish surface and formed filopodia. The cultured cells retained both granules and the ability to release amylase in response to beta-adrenergic and cholinergic agonists, even 48 h after dispersion. However, the total amount of amylase in the cells decreased rapidly from 24 to 48 h after dispersion. These results suggested that amylase synthesis was more damaged than the machinery for exocytosis during culture in vitro. VAMP2 gene fused with enhanced green fluorescence protein was transfected into the dispersed acinar cells, and VAMP2 protein was expressed and localized to amylase-containing granules, as normally seen for endogenous VAMP2 protein. This indicated that new granules were generated, and that protein sorting was functional. The cells cultured by this method maintained their functions for at least 48 h. They can be used for examining the effects of exogenous genes on parotid acinar cell functions, such as regulated exocytosis and the maturation of secretory granules.
Fujita-Yoshigaki
Junko
J
Department of Physiology, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-nishi, Matsudo, Chiba, 271-8587, Japan. yosigaki@mascat.nihon-u.ac.jp
Tagashira
Asako
A
Yoshigaki
Tomoyoshi
T
Furuyama
Shunsuke
S
Sugiya
Hiroshi
H
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000681:Amylases / Q000378:metabolism*; D000818:Animals; D016764:Cell Polarity; D002478:Cells, Cultured; D024042:Collagen Type I; D005089:Exocytosis; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D016210:Methacholine Chloride / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D010306:Parotid Gland / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D014162:Transfection; D050988:Vesicle-Associated Membrane Protein 2 / Q000096:biosynthesis / Q000235:genetics
D000318:Adrenergic beta-Agonists; D024042:Collagen Type I; D018721:Muscarinic Agonists; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2; C485184:enhanced green fluorescent protein; D016210:Methacholine Chloride; D049452:Green Fluorescent Proteins; D000681:Amylases; D007545:Isoproterenol