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J. Gierow, M.C. Edman, S.V. Andersson; Tyrosine Kinases and Tyrosine Phosphatases in Lacrimal Gland Intracellular Signaling . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4408.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Our laboratory has previously investigated the role of cholinergic and integrin signaling in regulated secretion of lacrimal gland cells (Andersson et al., Glycobiology, in press; Andersson et al., Ocular Surface 3:1, s43, 2005). The aim of the present study is to further investigate the intracellular signaling events involved, with special emphasis on tyrosine phosphorylation. Methods:Rabbit lacrimal gland acinar cells were prepared according to our standard procedure (Gierow et al., Am. J. Physiol. (Cell Physiology) 271: C1685, 1996) yielding single cells that were kept in a serum–free culture medium on culture plates coated with Matrigel for two days. Secretory capacity was measured after a pre–incubation for 30 min at 37°C w./w.o. tyrosine kinase and tyrosine phosphatase inhibitors followed by incubation at 37°C as inicated w./w.o. the presence of carbachol and monoclonal integrin subunit Ab:s. Basal secretion and regulated secretion was determined enzymatically as secreted beta–hexosaminidase activity. Cell lysates were analyzed by Western blotting using a primary mouse monoclonal Phosphotyrosine Ab (1 µg/ml) and a secondary goat anti–mouse Ab conjugated to HRP (1:10 000). Bands were visualized by ECL–Plus. Results:In concordance with our previous studies, carbachol and Ab:s against the α6 and ß1 integrin subunits resulted in a 7–9 fold increase in secretion. The presence of a general tyrosine kinase inhibitor (genistein) or a specific Src–kinase inhibitor (PP2) resulted in a 30–50% reduction of carbachol– and ß1–stimulated secretion, whereas the secretion induced by α6–Ab was unaffected. Ortovanadate, a general tyrosine phosphatase inhibitor, reduced basal, carbachol– and ß1–stimulated secretion by 50–70%, and α6–stimulated secretion by less than 20%. Western blots revealed 2 major bands (90 and 110 kD) in controls and in cells incubated with carbachol or integrin Ab:s. Carbachol–stimulation increased the staining of the 110 kD–band. Pre–incubation with vanadate introduced a 3rd band at approx. 115 kD, with the strongest staining in control cells. Conclusions: Our results indicate that tyrosine phosphorylation and dephosphorylation events play an important role in the secretory response elicited by carbachol and ß1–Ab in the rabbit lacrimals gland.
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