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Y. Wang, T. Nakamura, F. Yarber, D. Stevenson, J.E. Schechter4, M.D. Trousdale, S.F. Hamm–Alvarez, A.K. Mircheff; Prolactin Over–Expression Disrupts Merocrine Secretion in Lacrimal Acinar Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1970.
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© ARVO (1962-2015); The Authors (2016-present)
Studies by Ding and Schechter indicate that pregnancy is associated with decreased lacrimal gland fluid protein concentration and increased ocular surface disease. We have produced a vector (AdPRL) to efficiently express rabbit prolactin (PRL) in lacrimal acinar cells (LGCs) and found that over–expressed PRL is secreted into culture media in both constitutive– and stimulation–dependent pathways. A second vector (AdSynGFP) was used to transduce a chimeric secretory protein construct, syncollin–GFP, into PRL–over–expressing cells to test the hypothesis that excessive local levels of PRL alter the classic merocrine secretory pathway.
Primary LGCs were cultured for 2 days to form acinus–like structures, then infected with AdSynGFP or co–infected with AdPRL plus AdSynGFP or AdLacZ plus AdSynGFP, each with an MOI of 6. After culture for 18 hr, fresh medium with or without 100 µM carbachol (CCh) was added and the LGCs incubated an additional 20 min. Live cells were imaged by time–lapse confocal fluorescence and DIC microscopy. Duplicate cell samples were fixed with 4% paraformaldehyde and permeabilized with 0.2% triton X–100, then stained with rhodamine–phalloidin to label actin microfilaments. Amounts of syncollin–GFP released to the media were determined by Western blotting.
Excessive PRL had no obvious effect on the amounts of syncollin–GFP secreted. However, live cell imaging revealed that, while syncollin–GFP–containing vesicles were concentrated in the apical cytoplasm in singly–transduced– and AdLacZ+AdSynGFP–transduced cell preparations, they were distributed throughout the cytoplasm in AdPRL+AdSynGFP transduced cells. After CCh stimulation, syncollin–GFP–containing vesicles moved to the apical membrane (APM) in AdSynGFP– and AdLacZ+AdSynGFP–transduced cells, in contrast, this movement to APM was hindered, and the vesicles moved to the basal–lateral membranes (BLM) instead in AdPRL+AdSynGFP–transduced cells. Confocal imaging of actin microfilament networks underlying the APM and BLM in rhodamine–phalloidin labeled cells confirmed these phenomena.
PRL over–expression disrupts the classic merocrine secretory pathway and promotes aberrant mobilization of secretory vesicles to the BLM. We speculate that these functional changes might decrease the protein content of lacrimal gland fluid and increase the discharge of intracellular autoantigens to the underlying tissue space in physiological hyperprolactinemic states.
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