May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Adenoviral vector mediated over–expression of prolactin and its effect on apical secretory pathways in rabbit lacrimal acinar cells
Author Affiliations & Notes
  • Y. Wang
    Physiology and Biophysics,
    University of Sourthern California, Los Angeles, CA
  • T. Nakamura
    Physiology and Biophysics,
    University of Sourthern California, Los Angeles, CA
  • F. Yarber
    Pharmaceutical Science,
    University of Sourthern California, Los Angeles, CA
  • D. Stevenson
    Ophthalmology,
    University of Sourthern California, Los Angeles, CA
  • M. Trousdale
    Ophthalmology,
    University of Sourthern California, Los Angeles, CA
  • S.F. Hamm–Alvarez
    Pharmaceutical Science,
    University of Sourthern California, Los Angeles, CA
  • A.K. Mircheff
    Physiology and Biophysics,
    University of Sourthern California, Los Angeles, CA
  • Footnotes
    Commercial Relationships  Y. Wang, None; T. Nakamura, None; F. Yarber, None; D. Stevenson, None; M. Trousdale, None; S.F. Hamm–Alvarez, None; A.K. Mircheff, None.
  • Footnotes
    Support  EY13720, EY11386, ET05801, and DK48522
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3876. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Y. Wang, T. Nakamura, F. Yarber, D. Stevenson, M. Trousdale, S.F. Hamm–Alvarez, A.K. Mircheff; Adenoviral vector mediated over–expression of prolactin and its effect on apical secretory pathways in rabbit lacrimal acinar cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3876.

      Download citation file:


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

      ×
  • Supplements
Abstract

Abstract: : Purpose: Prolactin (PRL) is a pleiotropic pituitary hormone that shares many properties with cytokines. PRL is synthesized to a variable degree in extrapituitary tissues, including lacrimal epithelial cells. Some observations suggest that excessive PRL may be involved in dysfunctional tear syndromes. However, the effects of locally–produced PRL versus circulating PRL have not been established. The goal of this study was to assess the effect of PRL over–expression on the apical secretory pathway in lacrimal acinar cells ex vivo. Methods: cDNA for rabbit PRL was generously provided by Dr. Petridou. Adenovirus PRL (AdPRL) and HA–tagged PRL (AdPRL–HA) were constructed using the Adeno–X Expression System. Primary rabbit lacrimal acinar ceslls (LGCs) were cultured for 2 days under conditions favoring recontitution of acinus–like structures, then infected for 2 hrs with AdPRL, AdPRL–HA, AdGFP, or AdLacZ. After culture for 1–2 days more, LGCs were incubated an additional 15 min with or without 100 µM carbachol (CCh). PRL mRNA levels were evaluated by real–time RT–PCR (TaqMan). PRL immunoreactivity was determined by Western blotting using anti–rabbit PRL antibodies. PRL and markers for biosynthetic, secretory, and recycling compartments were evaluated by confocal immunofluorescence microscopy. Results: All Ad vectors resulted in highly efficient transduction as revealed by FACS and confocal fluorescence microscopy. Overexpression of PRL was confirmed by real–time PCR and Western blotting. CCh stimulation increased PRL secretion 20–fold. Both endogenous and transduced PRLs were distributed in a punctate pattern throughout the cytoplasm and showed partial colocalizations with rab6 and γ–adaptin (Golgi and TGN), rab11 (endosomes), and VAMP2 (recruitable secretory transport vesicles). PRL overexpression did not alter either the resting distributions of p150Glued and VAMP2 or their CCh–induced recruitment into the subapical cytoplasm. However, overexpression of PRL dispersed rab3D from mature, subapical secretory vesicles to the cytoplasm and recruited rab11 into the subapical cytoplasm. Conclusions: Ad constructs efficiently overexpress PRL in LGCs. PRL overexpression disrupts membrane compartments implicated in exocytosis and recycling of mature secretory vesicles but preserves the recruitable secretory transport vesicle pathway mediated by dynein and VAMP2.

Keywords: lacrimal gland • cornea: tears/tear film/dry eye • cell membrane/membrane specializations 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×