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G.V. Jerdeva, F.A. Yarber, S.R. da Costa, S.F. Hamm-Alvarez; Actin Filaments Participation in Stimulated Exocytosis in Primary Rabbit Lacrimal Acinar Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2518.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Although the apical actin network forms an apparent barrier to fusion of mature secretory vesicles at the apical membrane in lacrimal acini, evidence of its involvement in exocytosis in exocrine tissues is controversial. Here, we investigate the role of the actin cytoskeleton during stimulated apical secretion in lacrimal acinar cells. Methods: Lacrimal acini isolated from rabbit lacrimal gland and cultured for three days were treated with the actin destabilizing agent, cytochalasin D (CD, 5 µM, 60 min), or electroporated with the actin stabilizing agent, phalloidin (PH, 10 µM, 3-6 hrs recovery). Acini were fixed and processed for analysis of apical actin filaments and rab3D-enriched mature secretory vesicles by confocal fluorescence microscopy using appropriate primary and fluorescent secondary antibodies or affinity label. Basal and carbachol-stimulated (CCH, 100 µM) release of bulk protein was measured at time intervals from 0-30 min. Results: Confocal fluorescence microscopy revealed that CCH-stimulation caused an apparent decrease in the intensity of apical actin labeling relative to resting acini, as well as an increase in apparent actin-enriched vesicular structures the size of mature secretory vesicles at the apical membrane. Introduction of fluorescent PH confirmed an increase in apical actin filament content associated with PH electroporation. CD treatment disrupted basolateral actin while generating discontinuities but no substantial loss in the labeling intensity of apical actin. Analysis of rab3D-enriched mature secretory vesicle content by confocal fluorescence microscopy revealed that CCH stimulation promoted dispersal of almost all apical stores of this protein within 15 min. This effect was partly inhibited by PH but not by CD. PH treatment resulted in a significant (p<0.05) decrease in stimulated protein secretion at 30 min by >45%. In contrast, CD treatment significantly (p<0.05) increased stimulated protein release at 30 min by >230%. Analysis of the kinetics of stimulated protein release in CD-treated acini actually revealed a significant (p<0.05) increase in the earliest phase of secretion after 5 min of CCH, a time associated with discharge of rab3D-enriched vesicles. Conclusions: Increased actin disassembly (CD) enhances secretion while increased actin assembly (PH) impairs secretion, suggesting that actin filaments form a partial restrictive barrier for release of mature secretory vesicles in lacrimal gland. Support: NIH grant EY-11386. Commercial Relationship: none
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