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

• Published in: American Journal of Physiology: Cell Physiology Vol. 58; no. 4; p. C1052
• Main Authors: Jerdeva, Galina V, Yarber, Francie A, Trousdale, Melvin D, Rhodes, Christopher J
• Format: Journal Article
• Language: English
• Published: Bethesda American Physiological Society 01.10.2005
• Subjects: Cellular biology; Excretory system; Inhibitor drugs; Proteins; Rabbits; Signal transduction
• ISSN: 0363-6143; 1522-1563

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 microM, 2-15 min) significantly (P is less than or equal to 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 microM, 3 h), which inhibits PKC-{alpha}, -{beta}, -{delta}, and -{epsilon}, also elicited more potent inhibition of carbachol-stimulated secretion relative to Go-6976 (10 microM, 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. [PUBLICATION ABSTRACT]