May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Transduced Viral IL-10 Is Exocytosed in a Myosin-Dependent Manner From Lacrimal Acinar Secretory Vesicles in Response to Carbachol
Author Affiliations & Notes
  • J. Xie
    University of Southern California, Los Angeles, California
    Department of Pharmacology and Pharmaceutical Sciences,
  • R. Marchelletta
    University of Southern California, Los Angeles, California
    Department of Pharmacology and Pharmaceutical Sciences,
  • D. Stevenson
    University of Southern California, Los Angeles, California
    Department of Ophthalmology,
  • F. A. Yarber
    University of Southern California, Los Angeles, California
    Department of Pharmacology and Pharmaceutical Sciences,
  • S. F. Hamm-Alvarez
    University of Southern California, Los Angeles, California
    Department of Pharmacology and Pharmaceutical Sciences,
  • M. D. Trousdale
    University of Southern California, Los Angeles, California
    Department of Ophthalmology,
  • Footnotes
    Commercial Relationships J. Xie, None; R. Marchelletta, None; D. Stevenson, None; F.A. Yarber, None; S.F. Hamm-Alvarez, None; M.D. Trousdale, None.
  • Footnotes
    Support NIH Grants EY012689, EY011386 and EY03040
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1911. doi:
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      J. Xie, R. Marchelletta, D. Stevenson, F. A. Yarber, S. F. Hamm-Alvarez, M. D. Trousdale; Transduced Viral IL-10 Is Exocytosed in a Myosin-Dependent Manner From Lacrimal Acinar Secretory Vesicles in Response to Carbachol. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1911.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: We have previously shown that viral IL-10 (vIL-10) is exocytosed from transduced acinar epithelial cells from rabbit lacrimal gland in response to carbachol (CCH), a process likely to be targeted into the acinar cells’ regulated secretory pathway (IOVS 47: E-Abstract 1964, 2006). Here we sought to further characterize the intracellular trafficking and release pathways of vIL-10 from transduced acinar cells.

Methods:: Primary cultured rabbit lacrimal gland acinar cells (LGAC) were transduced with AdvIL-10 at a MOI of 5 for 24 hrs. The distribution of vIL-10 and that of other markers was assessed by confocal fluorescence microscopy. CCH (100 µM)-stimulated release of vIL-10 and the secretory product, ß-hexosaminidase, were measured at time intervals from 0-180 min using ELISA and biochemical assays, respectively. In some experiments, the sensitivity of vIL-10 localization and release to agents which influence the microtubule-dependent and actin-dependent transport processes were examined.

Results:: Confocal fluorescence microscopy revealed that in resting acini, vIL-10 was partially co-localized with biosynthetic but not endosomal markers. Secretion experiments showed a distinct delayed initial burst phase for CCH-stimulated vIL-10 release over 30 min. Disassembly of actin filaments with latrunculin B (LAT) resulted in significant (p≤0.05, n=5) enhancement of CCH-stimulated vIL-10 release while microtubule depolymerizing agent nocodazole had no effect. Myosin inhibitor ML-7 (p≤0.05, n=8) and overexpression of dominant negative myosin Vc with Ad-MyoVc-tail-GFP transduction (p≤0.05, n=10) both decreased CCH-stimulated vIL-10 secretion. Consistently, confocal fluorescence microscopy showed that CCH stimulation for 30 min reduced vIL-10 staining, especially that adjacent to the apical membrane. LAT pretreatment reduced vIL-10 staining in transduced cells stimulated by CCH while Ad-MyoVc-tail-GFP enhanced vIL-10 staining in AdvIL-10/Ad-MyoVc-tail-GFP - transduced cells. Moreover, considerably more co-localization of vIL-10 and MyoVc-tail-GFP was observed in the subapical region of doubly-transduced cells stimulated by CCH.

Conclusions:: The majority of vIL-10 transgene product is routed through the apical secretory pathway where it is sequestered in a subpopulation of mature secretory vesicles and released, in response to CCH, in a process that involves actin filaments and myosin motor proteins.

Keywords: lacrimal gland • gene transfer/gene therapy • cell membrane/membrane specializations 
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