April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Identification of Signaling Pathways Utilized by Leukotrienes D4 and E4 to Stimulate Rat Conjunctival Cell Secretion and Resolvins RvD1 and RvE1 to Block It
Author Affiliations & Notes
  • Darlene A. Dartt
    Schepens Eye Research Institute, Boston, Massachusetts
  • Robin R. Hodges
    Schepens Eye Research Institute, Boston, Massachusetts
  • Dayu Li
    Schepens Eye Research Institute, Boston, Massachusetts
  • Marie A. Shatos
    Schepens Eye Research Institute, Boston, Massachusetts
  • Charles N. Serhan
    Brigham and Womens Hospital, Boston, Massachusetts
  • Footnotes
    Commercial Relationships  Darlene A. Dartt, None; Robin R. Hodges, None; Dayu Li, None; Marie A. Shatos, None; Charles N. Serhan, None
  • Footnotes
    Support  NIH EY19470
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 4791. doi:https://doi.org/
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      Darlene A. Dartt, Robin R. Hodges, Dayu Li, Marie A. Shatos, Charles N. Serhan; Identification of Signaling Pathways Utilized by Leukotrienes D4 and E4 to Stimulate Rat Conjunctival Cell Secretion and Resolvins RvD1 and RvE1 to Block It. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4791. doi: https://doi.org/.

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

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Abstract

Purpose: : We previously showed that in rat conjunctival goblet cells the leukotrienes (LTs) LTD4 and LTE4 stimulate mucin secretion, which was inhibited by the proresolution compounds resolvins RvD1 and RvE1. This study was to determine the signaling pathways used by these LTs to stimulate secretion and the effects of RvD1 and RvE1 on these pathways.

Methods: : Immunohistochemistry. First passage goblet cells were incubated with antibodies against the RvD1 receptor, ChemR23, and the RvE1 receptor, GPR32. Measurement on Intracellular [Ca2+] ([Ca2+]i). Cultured goblet cells were incubated with fura-2 ester and stimulated with agonists alone or preincubated with RvD1 or RvE1. Fluorescent images of cells were analyzed with a digital fluorescence imaging system (InCyte Im2, Intracellular Imaging Inc.). Measurement of extracellular regulated kinase 1/2 (ERK) activity. Goblet cells were serum starved for 2 hrs and stimulated with LTD4 (10-11M) for 0-10 min or preincubated with RvD1 or RvE1 before stimulation with LTD4 (10-11M) for 5 min. Western blot was performed with antibodies directed against phosphorylated (active) ERK1/2 and total ERK2. The amount of phosphorylated ERK1/2 was standardized to the amount of total ERK2.

Results: : ChemR23 was found in a punctuate pattern in the cytosol while GPR32 was localized in a distinct area of the cytoplasm. LTD4 increased the [Ca2+]i a maximum increase of 104 ± 19 nM at 10-9M. LTE4 increased the [Ca2+]i a maximum of 514 ± 58 nM at 10-9 M. RvD1 completely attenuated the LTD4 and LTE4 increase in [Ca2+]i. RvE1 blocked the LTD4-stimulated increase in [Ca2+]i by 70% and LTE4 stimulated [Ca2+]i increase by 85%. When phosphoERK1/2 activity was measured, LTD4 (10-11M) activated ERK1/2 2.0 ± 0.3 fold. RvD1 completely abolished the activation of ERK1/2. RvE1 decreased it by 80%.

Conclusions: : Thus RvD1 and RvE1 each block LTD4 and LTE4 stimulated goblet cell secretion by preventing the increase in the [Ca2+]i and the activation of ERK1/2, the two major mechanisms by which conjunctival goblet cell secretion is stimulated.

Keywords: conjunctiva • signal transduction • inflammation 
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