TRPV4 in porcine lens epithelium regulates hemichannel-mediated ATP release and Na-K-ATPase activity

Abstract

In several tissues, transient receptor potential vanilloid 4 (TRPV4) channels are involved in the response to hyposmotic challenge. Here we report TRPV4 protein in porcine lens epithelium and show that TRPV4 activation is an important step in the response of the lens to hyposmotic stress. Hyposmotic solution (200 mosM) elicited ATP release from intact lenses and TRPV4 antagonists HC 067047 and RN 1734 prevented the release. In isosmotic solution, the TRPV4 agonist GSK1016790A (GSK) elicited ATP release. When propidium iodide (PI) (MW 668) was present in the bathing medium, GSK and hyposmotic solution both increased PI entry into the epithelium of intact lenses. Increased PI uptake and ATP release in response to GSK and hyposmotic solution were abolished by a mixture of agents that block connexin and pannexin hemichannels, 18α-glycyrrhetinic acid and probenecid. Increased Na-K-ATPase activity occurred in the epithelium of lenses exposed to GSK and 18α-glycyrrhetinic acid + probenecid prevented the response. Hyposmotic solution caused activation of Src family kinase and increased Na-K-ATPase activity in the lens epithelium and TRPV4 antagonists prevented the response. Ionomycin, which is known to increase cytoplasmic calcium, elicited ATP release, the magnitude of which was no greater when lenses were exposed simultaneously to ionomycin and hyposmotic solution. Ionomycin-induced ATP release was significantly reduced in calcium-free medium. TRPV4-mediated calcium entry was examined in Fura-2-loaded cultured lens epithelium. Hyposmotic solution and GSK both increased cytoplasmic calcium that was prevented by TRPV4 antagonists. The cytoplasmic calcium rise in response to hyposmotic solution or GSK was abolished when calcium was removed from the bathing solution. The findings are consistent with hyposmotic shock-induced TRPV4 channel activation which triggers hemichannel-mediated ATP release. The results point to TRPV4-mediated calcium entry that causes a cytoplasmic calcium increase which is an essential early step in the mechanism used by the lens to sense and respond to hyposmotic stress.

Mechanosensitive release of adenosine 5′‐triphosphate through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: A mechanism for purinergic involvement in chronic strain

“…Whether the association between pannexins and actin in Figure 5 indicates a more direct role in mechanosensitivity or is merely a reflection of the cultured condition, as found in cortical astrocytes (Oberheim et al 2009), remains to be determined. An upstream role for TRPV4 has been implicated in the swelling-activated ATP release from pannexins in lens (Shahidullah et al 2012) and airway epithelial cells (Seminario-Vidal et al 2011). However, experiments to determine a role of TRPV4 in the present study were inconclusive due to the substantial effect of inhibitor HC067047 on the luciferase standard curve (not shown).…”

TRPV4 activation in mouse submandibular gland modulates Ca2+influx and salivation

“…GSK1016790A induces a Ca 2ϩ increase in several different cell types at nanomolar levels (13,14,33). For example, in TRPV4-transfected HeLa cells, GSK1016790A produces a dose-dependent Ca 2ϩ increase with an EC 50 of 3.3 nM (14).…”

Purines in the eye: Recent evidence for the physiological and pathological role of purines in the RPE, retinal neurons, astrocytes, Müller cells, lens, trabecular meshwork, cornea and lacrimal gland

“…With a molecular weight of 668, PI would be excluded by most channels but not hemichannels. It is interesting that both increased PI uptake and ATP release in hyposmotic solution were blocked by RN 1734, a very different channel blocker that interferes with TRPV4 channels (Shahidullah et al, 2012b). Moreover, it was found that PI entry into the lens epithelium was increased by more than twofold in lenses exposed to a TRPV4 agonist GSK1016790A (GSK).…”