Effects of acetylcholine chloride on intracellular calcium concentration of cultured sweat gland epithelial cells.
In order to isolate and culture the sweat gland epithelial cells in vitro and to study the effects of acetylcholine (ACh) on intracellular calcium concentration ([Ca(2+)](i)) of cultured sweat gland epithelial cells, the following methods were used. First, repeated shearing and neutral red staining made the sweat glands pop out from subcutaneous tissues. Then, transferpettor was used to pick up the glands, which were cultured in Epilife after type II collagenase digestion. The molecular characterization of primary cultured sweat gland epithelial cells was shown by immunocytochemistry. The [Ca(2+)](i) was examined by confocal laser scanning microscopy (CLSM) with the Ca(2+)-sensitive dye Fura 3-AM, when ACh was added to the primary cells and the first passage cells. In the results, the established method yielded comparatively more sweat glands, and the primary and first passage epithelial cells developed well in Epilife. The primary epithelial cells were positive to anti-EMA, anti-CK and anti-CK7. After the ACh was added, when the medium with high calcium (2 mmol/L) was applied, the calcium channel of both primary and first passage cells opened and significant [Ca(2+)](I )increase was observed; when the medium with no calcium was applied, no significant [Ca(2+)](i )increase was observed. So, it is a good method to isolate sweat glands by repeated shearing and transferpettor picking, and the culture mediums of keratinocytes, like Epilife, can be used to culture the sweat glands epithelial cells. In both the cultured primary and first passage cells, when stimulated by ACh, the calcium channel opened, which induced an increase in [Ca(2+)](i), similar to the cells in vivo.
Lei
Xia
X
Department of Dermatology, Daping Hospital, The Third Military Medical University, Chongqing, China. leixia1979@sina.com
Wu
Jinjin
J
Lu
Yuangang
Y
Zhu
Tangyou
T
D000109:Acetylcholine / Q000031:analogs & derivatives / Q000494:pharmacology*; D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation / Q000276:immunology; D002118:Calcium / Q000819:agonists / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002469:Cell Separation; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D013545:Sweat Glands / Q000166:cytology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D015220:Calcium Channels; D000109:Acetylcholine; D002118:Calcium