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R.R. Marchelletta, Y. Wang, L. Qian, C.M. Rose, A.K. Mircheff, S.F. Hamm-Alvarez; Stimulated Trafficking of Prolactin to the Apical Plasma Membrane in Primary Rabbit Lacrimal Acini is Facilitated by Microtubules and Cytoplasmic Dynein . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3779.
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Purpose: Maintenance of lacrimal gland function relies upon its exposure to a host of effectors like growth factors and hormones such as prolactin (PRL). Interestingly, while lacrimal acini express prolactin receptors responsive to extracellular PRL, they also contain intracellular PRL which is released directly into tear fluid. Excessive PRL from endocrine or autocrine stores can alter lacrimal gland function, contributing to the development of autoimmune diseases such as Sjögren's syndrome. Here we explore the mechanisms responsible for stimulated traffic of PRL to the apical plasma membrane in lacrimal acini. Methods: For confocal fluorescence microscopy, resting and carbachol-stimulated (CCH, 100 µM, 15 min) rabbit lacrimal acini cultured on Matrigel-coated coverslips without and with exposure to nocodazole (33 µM, 60 min) or adenoviral constructs containing the dynein inhibitor, dynamitin (Ad-Dynt), or the reporter gene, ß-galactosidase (Ad-LacZ) were fixed and processed using appropriate primary and fluorescent secondary antibodies or affinity label. PRL association with subcellular membranes was assessed by separation over sorbitol density gradients and phase partitioning into an aqueous dextran - polyethyleneglycol two-phase system followed by Western blotting and/or activity measurements. Results: Confocal fluorescence microscopy revealed that PRL exhibited a filamentous pattern in resting acini that was colocalized with microtubules. CCH stimulation resulted in its accumulation beneath the apical plasma membrane and its apparent colocalization with dynein constituents and VAMP2, components which delineate recruitable secretory vesicles. Nocodazole and Ad-Dynt but not Ad-LacZ impaired CCH-stimulated accumulation of PRL beneath the apical plasma membrane. Further, sorbitol density gradient analysis and phase partitioning revealed substantial overlap between dynein constituents and PRL in CCH-stimulated acini. Conclusions: CCH-stimulated movement of intracellular PRL to the apical membrane is facilitated by microtubules and dynein. Since excessive PRL can alter lacrimal gland function while potentiating autoimmunity via its cytokine actions, elucidation of the mechanisms responsible for its stimulated release is important in our understanding of the etiology of Sjögren's syndrome.
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