Mechanism for carbachol-induced secretion of lacritin in cultured monkey lacrimal acinar cells

Ayumi Morimoto-Tochigi, Ryan D. Walkup, Emi Nakajima, Thomas (Tom) Shearer, Mitsuyoshi Azuma

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

PURPOSE. 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 storeoperated 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.

Original languageEnglish (US)
Pages (from-to)4395-4406
Number of pages12
JournalInvestigative Ophthalmology and Visual Science
Volume51
Issue number9
DOIs
StatePublished - Sep 2010

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Acinar Cells
Carbachol
Tears
Haplorhini
Mitogen-Activated Protein Kinase 3
Mitogen-Activated Protein Kinase 1
Lacrimal Apparatus
Primates
Immunohistochemistry
Calcium
Lipocalins
Cholinergic Agonists
Proteins
Lactoferrin
Mitogen-Activated Protein Kinase Kinases
Calcium Channels
Messenger RNA
Genes

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience
  • Medicine(all)

Cite this

Mechanism for carbachol-induced secretion of lacritin in cultured monkey lacrimal acinar cells. / Morimoto-Tochigi, Ayumi; Walkup, Ryan D.; Nakajima, Emi; Shearer, Thomas (Tom); Azuma, Mitsuyoshi.

In: Investigative Ophthalmology and Visual Science, Vol. 51, No. 9, 09.2010, p. 4395-4406.

Research output: Contribution to journalArticle

Morimoto-Tochigi, Ayumi ; Walkup, Ryan D. ; Nakajima, Emi ; Shearer, Thomas (Tom) ; Azuma, Mitsuyoshi. / Mechanism for carbachol-induced secretion of lacritin in cultured monkey lacrimal acinar cells. In: Investigative Ophthalmology and Visual Science. 2010 ; Vol. 51, No. 9. pp. 4395-4406.
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T1 - Mechanism for carbachol-induced secretion of lacritin in cultured monkey lacrimal acinar cells

AU - Morimoto-Tochigi, Ayumi

AU - Walkup, Ryan D.

AU - Nakajima, Emi

AU - Shearer, Thomas (Tom)

AU - Azuma, Mitsuyoshi

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N2 - PURPOSE. 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 storeoperated 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.

AB - PURPOSE. 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 storeoperated 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.

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