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Ting Wei, Mohammad Shahidullah, Nicholas A Delamere, Amritlal Mandal; Porcine NPE releases ATP through interaction of TRPV4 and hemichannels. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1221.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies showed the presence of connexin 43, connexin 50, pannexin 1 proteins and hemichannel (HC) activity in the nonpigmented ciliary epithelium (NPE) of the eye. In the lens mechanosensitive Transient Receptor Potential Vanilloid 4 (TRPV4)-mediated calcium entry stimulates hemichannel activity. The aim here is to test for TRPV4 expression in the NPE and examine its functional interaction with HCs.
Immunohistochemistry was used to probe for TRPV4 in porcine ciliary body. Propidium iodide (PI) uptake and ATP release by primary cultured NPE was used to assess HC opening. PI and ATP are large molecules (MW 668 and 507, respectively) that pass through open hemichannels but not the plasma membrane. ATP in the bathing medium was measured by luciferase luminometry and PI uptake by fluorimetry.
TRPV4 was detected in the NPE, mainly at the basolateral surface. Cultured NPE cells displayed an increase in PI uptake when exposed to a TRPV4 agonist GSK 1016790A (10 nM) or to hyposmotic solution (200 mOsm) (Control 4.65 ± 0.24 vs GSK 10.26 ± 0.32 and Hypo 8.26 ± 0.42 fluorescence units per mg protein; n=6, p<0.001). PI uptake also was increased in NPE cells cultured on a flexible membrane and subjected to a cyclic stretch (10%, 0.5 Hz) (Control 1.5 ± 0.03 vs stretch 2.5 ± 0.10; n=6, p<0.001). The increase in PI uptake caused by hyposmotic solution or by cyclic stretch was abolished by a selective TRPV4 antagonist HC 067047 (10 μM). Hyposmotic solution increased ATP release 1.12 ± 0.24 to 2.81± 0.27 (n=12, p<0.001) while cyclic stretch increased ATP release from 0.20 ± 0.03 to 0.40 ± 0.03 pmoles/mg protein (n=6, p<0.001). ATP release responses caused by hypoosmotic solution and cyclic stretch were inhibited by HC 067047. ATP release caused by hyposmotic solution also was blocked by connexin inhibitory peptide, Gap-27 (200 μM).
TRPV4 channels are located on the basolateral (aqueous humor-facing) surface of the NPE. Prevention of ATP release and PI uptake by HC 067047 and Gap-27 suggests interaction of TRPV4 with connexin HCs. Connexin 50 is implicated in the HC response but we cannot rule out involvement of pannexin and other connexins.
This is a 2020 ARVO Annual Meeting abstract.
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