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G.V. Jerdeva, F.A. Yarber, M.D. Trousdale, D.A. Dartt, S.F. Hamm–Alvarez; PKC Function in Apical Exocytosis in Rabbit Lacrimal Acinar Cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3863.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Stimulation–induced exocytosis of secretory vesicles in lacrimal acinar cells (LAC) is accompanied by local actin filament remodeling. The protein kinase C isoform, PKCε, activated by cholinergic and α1–adrenergic stimulation, has a unique actin–binding motif. Here we test whether PKCε mediates actin reorganization during exocytosis. Methods: Endogenous PKCε localization and responsiveness to secretagogues was evaluated in 3 day cultures of rabbit LAC using confocal fluorescence microscopy and sequential detergent extractions. 2 day cultures of rabbit LAC were exposed to adenovirus containing dominant negative PKCε (DN PKCε) or GFP for 4 h (MOI 5–10). After overnight recovery, PKCε and actin organization and responsiveness to secretagogues were evaluated by confocal fluorescence microscopy in parallel with basal and stimulated protein secretion. Secretagogues included carbachol (CCH, 100 µM), phenylephrine (PHE, 100 µM) and phorbol myristate acetate (PMA,1 µM). Results: Sequential detergent extraction of resting acini revealed that 42 + 4 % of total PKCε was recovered in saponin–soluble fractions with the remainder distributed between Triton X–100 and SDS–soluble fractions. PMA significantly (p≤0.05, n=3) depleted saponin–soluble PKCε to 2 + 2 % of total PKCε while significantly increasing its recovery in Triton X–100 and SDS–soluble pools. Confocal fluorescence microscopy revealed PKCε immunoreactivity localized at the apical membrane and with apparent actin–coated fusing secretory vesicles in CCH– or PMA–stimulated acini, consistent with its enrichment with actin and membranes in stimulated acini suggested by detergent extraction. Comparable analysis of acini transduced with DN PKCε revealed that PKCε association with actin filaments in both resting and stimulated acini was greater or equal to the amount of endogenous PKCε recovered with actin in stimulated, non–transduced acini, an effect associated with altered acinar shape and accumulation of apical actin. DN PKCε significantly (p≤0.05) reduced CCH–stimulated protein release by 34% at 30 min (n=7) while Ad–GFP had no effect. Preliminary findings showed that DN PKCε exerted a greater inhibitory effect on secretion elicited by the PKCε–selective agonist, PHE, reducing protein release by 84% (n=2). Conclusions: PKCε is a likely candidate for apical actin remodeling during secretory vesicle exocytosis.
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