TRPV4 channels regulate the inflow pathway in the anterior eye

Abstract

Purpose: Aqueous humor dynamics is regulated by balanced production in the ciliary body and removal through the primary (“conventional”) and secondary outflow pathways. Fluid production is highly influenced by osmoregulatory mechanisms. To determine the molecular mechanism regulating inflow, we examined the expression and function of the osmosensory/mechanosensitive TRPV4 channels in the anterior eye.

Methods: The ciliary body was dissected from the mouse eye and incubated with agmatine (AGB, a cation influx marker). The tissue was exposed to selective TRP channel agonists, fixed and immunostained for AGB. Alternatively, epithelial cells were loaded with the calcium indicator dye fura-2 and stimulated with TRP-effective compounds; calcium concentration [Ca2+]i was determined using high-resolution optical imaging. The effect of TRP channel agonists in vivo was determined using high-resolution manganese-enhanced resonance imaging (MEMRI). Eye sections were processed for hematoxylin and eosin, apoptosis markers, metabolic markers, and EM ultrastructure.

Results: TRPV4 immunoreactivity was observed was expressed in the pars plicata of the fluid producing, non-pigmented epithelial (NPE) cells, but was absent from pigmented epithelial cells of the ciliary body. Hypotonic stimuli induced dose-dependent increases in NPE cell volume, which in turn was associated with entry of cations into the ciliary body. Both swelling response and hypotonicity-induced Ca signals were TRPV4-dependent. Accordingly, selective TRPV4 agonists such as GSK1016790A increased [Ca2+]i in NPE cells, augmented AGB immunoreactivity, and increased manganese uptake via calcium channels into the ciliary body in vivo. The extent of hypoosmotic swelling was reduced by BAPTA. Importantly HTS-induced changes in volume regulation, kinetics of hypotonic induced [Ca²⁺]_i signals, and calcium signal amplitude to GSK were compromised in mice lacking aquaporin 4 (AQP4) channels.

Conclusions: Our data provide molecular and physiological evidence that TRPV4 mediates osmosensation in the ciliary body, possibly as part of a macromolecular complex that includes the water channel AQP4. Thus, these findings suggest a novel mechanism that regulates inflow pathway in the anterior eye by balancing the bidirectional water transport and ion intake in NPE cells. By sensing NPE volume and adjusting aqueous secretion, TRPV4 is proposed to represent a linchpin in the regulation of intraocular pressure.