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

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