Dominant negative PKCɛ impairs apical actin remodeling in parallel with inhibition of carbachol-stimulated secretion in rabbit lacrimal acini

Here we investigate the involvement of PKCɛ in apical actin remodeling in carbachol-stimulated exocytosis in reconstituted rabbit lacrimal acinar cells. Lacrimal acinar PKCɛ 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 PKCɛ recovery with actin filaments in two distinct biochemical assays, while confocal fluorescence microscopy showed a significant increase in PKCɛ association with apical actin in stimulated acini as evidenced by quantitative colocalization analysis. Overexpression of dominant negative (DN) PKCɛ 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 β-hexosaminidase. The chemical inhibitor GF 109203X (10 μM, 3 hrs) which inhibits PKCα, β, δ and ɛ, also elicited more potent inhibition of carbachol-stimulated secretion relative to Go 6976 (10 μM, 3 hrs) which inhibits only PKCα and β. 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-PKCɛ significantly inhibited carbachol-stimulated release of syncollin-GFP. Carbachol also increased the recovery of secretory component in culture medium while Ad-DN-PKCɛ transduction suppressed its carbachol-stimulated release. We propose that DN-PKCɛ alters lacrimal acinar apical actin remodeling, leading to inhibition of stimulated exocytosis and transcytosis.