June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Characterization of TRPV4 expression & function in the ciliary body & trabecular meshwork
Author Affiliations & Notes
  • Amber Frye
    Ophthalmology & Visual Sciences, Moran Eye Institute, University of Utah School of Medicine, Salt Lake City, UT
  • Daniel Ryskamp
    Ophthalmology & Visual Sciences, Moran Eye Institute, University of Utah School of Medicine, Salt Lake City, UT
    Interdepartmental Program in Neuroscience, University of Utah, Salt Lake City, UT
  • Peter Barabas
    Ophthalmology & Visual Sciences, Moran Eye Institute, University of Utah School of Medicine, Salt Lake City, UT
  • Tunde Molnar
    Ophthalmology & Visual Sciences, Moran Eye Institute, University of Utah School of Medicine, Salt Lake City, UT
  • David Krizaj
    Ophthalmology & Visual Sciences, Moran Eye Institute, University of Utah School of Medicine, Salt Lake City, UT
    Interdepartmental Program in Neuroscience, University of Utah, Salt Lake City, UT
  • Footnotes
    Commercial Relationships Amber Frye, None; Daniel Ryskamp, None; Peter Barabas, None; Tunde Molnar, None; David Krizaj, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1974. doi:
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      Amber Frye, Daniel Ryskamp, Peter Barabas, Tunde Molnar, David Krizaj; Characterization of TRPV4 expression & function in the ciliary body & trabecular meshwork. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1974.

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

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Abstract

Purpose: Aqueous humor dynamics is regulated by balanced production in the ciliary body & removal through the primary (“conventional”) & secondary outflow pathways. Regulation of the volume of trabecular meshwork (TM) cells thus directly impacts the outflow facility & affects the amplitude of intraocular pressure. To determine the molecular mechanism regulating the outflow, we examined the expression & function of the osmosensory/mechanosensitive TRPV4 cation channel in the anterior chamber (AC) of the eye.

Methods: Cryosections from C57BL6 mouse eyes & human punches from trabeculectomies were immunostained with a validated TRPV4 antibody (Ryskamp et al., 2011) & colabeled with the TM marker aquaporin 1. Nuclei were labeled with propidium iodide & images were acquired with a confocal microscope. Alternatively, TM tissues were loaded with the calcium indicator dye fura-2 & the calcium-insensitive cell volume indicator calcein. Calcium levels [Ca2+]i and cell volume were determined using high-resolution optical imaging. To functionally map TRPV4 expression, eye sections were incubated with 100 nM GSK1016790A (GSK), a selective TRPV4 agonist, in the presence of extracellular agmatine (AGB 5 mM, a cation influx marker) and/or the TRPV4 antagonist HC067047 (HC 1 µM). Ocular slices were then fixed & immunostained for AGB. To assess the impact of maximal TRPV4 activation on histology, 75 µM GSK was injected into the AC. Eye slices were processed for H&E, apoptosis markers, metabolic markers, & EM ultrastructure.

Results: TRPV4 immunoreactivity was observed confirming its expression in the pars plicata of the fluid producing, non-pigmented epithelial cells lining the ciliary processes, but was absent from the pigmented epithelial cells of the ciliary body. Both mouse & human TM were labeled by the TRPV4 antibody. The functional presence of TRPV4 in these cells was confirmed by GSK- & HC-dependent changes in [Ca2+]i & AGB influx. In contrast to the pronounced proapoptotic effects of GSK in retinal cells, saturating TRPV4 activation induced minimal apoptosis & histological changes within the ciliary body & TM cells.

Conclusions: Our data provide molecular & physiological evidence of TRPV4 localization in the ciliary body, trabecular meshwork & the posterior eye and suggest a novel mechanism that could potentially regulate the volume of cells within the conventional outflow pathway.

Keywords: 427 aqueous • 455 ciliary body • 735 trabecular meshwork  
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