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Am J Physiol Cell Physiol (June 1, 2005). doi:10.1152/ajpcell.00546.2004
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Submitted on November 9, 2004
Accepted on May 25, 2005

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

Galina V Jerdeva1, Francie A Yarber1, Melvin D Trousdale2, Christopher J Rhodes3, Curtis T Okamoto1, Darlene A Dartt4, and Sarah F Hamm-Alvarez5*

1 Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, USA
2 Ophthalmology, University of Southern California, Los Angeles, CA, USA
3 Pacific Northwest Research Institute, Seattle, WA, USA
4 Schepens Eye Research Institute, Boston, MA, USA
5 Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, USA; Ophthalmology, University of Southern California, Los Angeles, CA, USA; Physiology and Biophysics, University of Southern California, Los Angeles, CA, USA

* To whom correspondence should be addressed. E-mail: shalvar{at}usc.edu.

Here we investigate the involvement of PKC{epsilon} in apical actin remodeling in carbachol-stimulated exocytosis in reconstituted rabbit lacrimal acinar cells. Lacrimal acinar PKC{epsilon} co-sedimented 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, while 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 using 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 hrs) which inhibits PKC{alpha}, {beta},{delta} and {epsilon}, also elicited more potent inhibition of carbachol-stimulated secretion relative to Go 6976 (10 µM, 3 hrs) which inhibits only PKC {alpha}and {beta}. Transduction of lacrimal acini with Ad encoding syncollin-GFP resulted in labeling of secretory vesicles that were discharged in response to carbachol stimulation while co-transduction 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 while 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.




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