Mechanism for carbachol-induced secretion of lacritin in cultured monkey lacrimal acinar cells.
OBJECTIVE
Lacritin protein is highly expressed in the lacrimal gland, secreted into tear fluid, and detected only in primates. The mechanism for lacritin secretion has not been fully investigated, because a system for culturing primate lacrimal acinar cells had not been established. The purposes of the present study were (1) to develop a procedure to culture lacrimal acinar cells from monkey and (2) to determine the mechanism for the secretion of lacritin in the culture system.


METHODS
Acinar cells from monkey lacrimal gland were cultured and characterized. Lacritin and other proteins were detected by immunohistochemistry, immunocytochemistry, and immunoblot analysis. Secreted proteins were also detected in the medium from stimulated acinar cells. mRNAs were determined by microarray and qPCR. Intracellular calcium levels were measured by calcium-4 assay.


RESULTS
Acinar cells cultured for 1 day contained adequate amounts of lacritin, lactoferrin, and lipocalin for use in lacritin secretion studies. The cholinergic agonist carbachol (Cch) stimulated the secretion of lacritin and increased intracellular Ca2+. Cch-induced lacritin secretion was inhibited by the store-operated calcium (SOC) channel inhibitor YM58483 and the PKC inhibitors GF109203 and Ro-32-0432. Cch-induced lacritin secretion was not inhibited by MAPKK inhibitor U0126, although p42/p44 MAPK was phosphorylated. Cch also enhanced gene transcription, which was inhibited by U0126, GF109203, and calcium chelators.


CONCLUSIONS
Successful culture of monkey lacrimal acinar cells showed that, among the prevalent tear proteins, the secretion of lacritin involved the PKC/Ca2+ pathway, not the p42/p44 MAPK pathway. Induction of transcription by Cch involved the independent p42/p44 MAPK and PKC pathways.
Morimoto-Tochigi
Ayumi
A
Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Beaverton, Oregon 97006, USA.
Walkup
Ryan D
RD
Nakajima
Emi
E
Shearer
Thomas R
TR
Azuma
Mitsuyoshi
M
D000813:Anilides / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D007211:Indoles / Q000494:pharmacology; D007765:Lacrimal Apparatus / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D008253:Macaca mulatta; D008301:Maleimides / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011758:Pyrroles / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013830:Thiadiazoles / Q000494:pharmacology
C476308:4-methyl-4'-(3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl)-1,2,3-thiadiazole-5-carboxanilide; D000813:Anilides; D018679:Cholinergic Agonists; D004791:Enzyme Inhibitors; D005136:Eye Proteins; D006023:Glycoproteins; D007211:Indoles; D008301:Maleimides; D011758:Pyrroles; D012333:RNA, Messenger; D013830:Thiadiazoles; C042434:tear proteins; C085989:Ro 32-0432; D002217:Carbachol; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C070515:bisindolylmaleimide I; D002118:Calcium