Dominant-negative PKC-epsilon impairs apical actin remodeling in parallel with inhibition of carbachol-stimulated secretion in rabbit lacrimal acini.
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 < or = 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 Gö-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.
Jerdeva
Galina V
GV
Department of Pharmaceutical Sciences, University of Southern California School of Pharmacy, 1985 Zonal Ave., Los Angeles, California 90033, USA.
Yarber
Francie A
FA
Trousdale
Melvin D
MD
Rhodes
Christopher J
CJ
Okamoto
Curtis T
CT
Dartt
Darlene A
DA
Hamm-Alvarez
Sarah F
SF
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology; D005260:Female; D007527:Isoenzymes; D007765:Lacrimal Apparatus / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D011817:Rabbits
D000199:Actins; D002352:Carrier Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; C107454:Sycn protein, rat; D002217:Carbachol; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon