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