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Am J Physiol Cell Physiol 289: C1052-C1068, 2005. First published June 1, 2005; doi:10.1152/ajpcell.00546.2004
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Protein and Vesicle Trafficking, Cytoskeleton

Dominant-negative PKC-{epsilon} impairs apical actin remodeling in parallel with inhibition of carbachol-stimulated secretion in rabbit lacrimal acini

Galina V. Jerdeva,1 Francie A. Yarber,1 Melvin D. Trousdale,2 Christopher J. Rhodes,4 Curtis T. Okamoto,1 Darlene A. Dartt,5 and Sarah F. Hamm-Alvarez1,2,3

Departments of 1Pharmaceutical Sciences, 2Ophthalmology, and 3Physiology and Biophysics, University of Southern California, Los Angeles, California; 4Pacific Northwest Research Institute, Seattle, Washington; and 5Schepens Eye Research Institute, Boston, Massachusetts

Submitted 9 November 2004 ; accepted in final form 25 May 2005

We investigated the involvement of PKC-{epsilon} in apical actin remodeling in carbachol-stimulated exocytosis in reconstituted rabbit lacrimal acinar cells. Lacrimal acinar PKC-{epsilon} cosedimented with actin filaments in an actin filament binding assay. Stimulation of acini with carbachol (100 µM, 2–15 min) significantly (P ≤ 0.05) increased PKC-{epsilon} recovery with actin filaments in two distinct biochemical assays, and confocal fluorescence microscopy showed a significant increase in PKC-{epsilon} association with apical actin in stimulated acini as evidenced by quantitative colocalization analysis. Overexpression of dominant-negative (DN) PKC-{epsilon} in lacrimal acini with replication-defective adenovirus (Ad) resulted in profound alterations in apical and basolateral actin filaments while significantly inhibiting carbachol-stimulated secretion of bulk protein and {beta}-hexosaminidase. The chemical inhibitor GF-109203X (10 µM, 3 h), which inhibits PKC-{alpha}, -{beta}, -{delta}, and -{epsilon}, also elicited more potent inhibition of carbachol-stimulated secretion relative to Gö-6976 (10 µM, 3 h), which inhibits only PKC-{alpha} and -{beta}. Transduction of lacrimal acini with Ad encoding syncollin-green fluorescent protein (GFP) resulted in labeling of secretory vesicles that were discharged in response to carbachol stimulation, whereas cotransduction of acini with Ad-DN-PKC-{epsilon} significantly inhibited carbachol-stimulated release of syncollin-GFP. Carbachol also increased the recovery of secretory component in culture medium, whereas Ad-DN-PKC-{epsilon} transduction suppressed its carbachol-stimulated release. We propose that DN-PKC-{epsilon} alters lacrimal acinar apical actin remodeling, leading to inhibition of stimulated exocytosis and transcytosis.

lacrimal gland; acinar epithelial cell; exocytosis; polymeric immunoglobulin A receptor


Address for reprint requests and other correspondence: S. F. Hamm-Alvarez, Dept. of Pharmaceutical Sciences, USC School of Pharmacy, 1985 Zonal Ave., Los Angeles, CA 90033 (e-mail: shalvar{at}usc.edu)




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