Abstract
Purpose:
Recently we reported on ATP release by lenses subjected to swelling by exposure to hyposmotic solution. While the mechanism of ATP release appeared to involve connexin/pannexin hemichannels, TRPV4 channel activation was a required step. TRPV4 may act as a mechanosensor that detects lens swelling and initiates a response. Here, we report on studies to further examine TRPV4-dependent responses and determine where TRPV4 protein is expressed in the lens.
Methods:
Porcine lens epithelium and fibers were separated and divided into various regions. TRPV4 protein was analyzed by Western blot analysis. By measuring fluorescence, propidium iodide (PI) uptake was examined as an indicator of hemichannel opening.
Results:
Consistent with swelling-induced hemichannel opening, PI uptake was increased in the epithelium of intact lenses exposed to hyposmotic solution. The TRPV4 antagonist HC067047 prevented the response. TRPV4 protein was detected in the equatorial region of the lens epithelium but was comparatively sparse in the anterior epithelium. TRPV4 was not detected in equatorial, anterior or posterior fibers. In an attempt to cause lens swelling by a maneuver that does not directly involve the epithelium, intact lenses were briefly subjected to selective freeze damage to fibers at the posterior lens pole. After 30 min, PI uptake was increased in the epithelium. HC067047 suppressed the PI uptake response to freeze damage.
Conclusions:
TRPV4 is expressed only in a small fraction of lens cells, mainly epithelial cells near the equator. In spite of such limited distribution, TRPV4 seems to play an important role in hemichannel opening that occurs even when the lens responds to damage that occurs in a remote region of the fiber mass.
Keywords: 569 ion channels •
532 gap junctions/coupling •
448 cell membrane/membrane specializations