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Differential volume regulation and calcium signaling in two ciliary body cell types is subserved by TRPV4 channels

Research paper by Andrew O. Jo, Monika Lakk, Amber M. Frye, Tam T. T. Phuong, Sarah N. Redmon, Robin Roberts, Bruce A. Berkowitz, Oleg Yarishkin, David Križaj

Indexed on: 23 Mar '16Published on: 22 Mar '16Published in: PNAS



Abstract

Fluid secretion by the ciliary body plays a critical and irreplaceable function in vertebrate vision by providing nutritive support to the cornea and lens, and by maintaining intraocular pressure. Here, we identify TRPV4 (transient receptor potential vanilloid isoform 4) channels as key osmosensors in nonpigmented epithelial (NPE) cells of the mouse ciliary body. Hypotonic swelling and the selective agonist GSK1016790A (EC50 ∼33 nM) induced sustained transmembrane cation currents and cytosolic <mml:math><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msup><mml:mrow><mml:mtext>Ca</mml:mtext></mml:mrow><mml:mrow><mml:mn>2</mml:mn><mml:mi mathvariant="normal">+</mml:mi></mml:mrow></mml:msup></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi mathvariant="normal">i</mml:mi></mml:msub></mml:mrow></mml:math>[Ca2+]i elevations in dissociated and intact NPE cells. Swelling had no effect on <mml:math><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msup><mml:mrow><mml:mtext>Ca</mml:mtext></mml:mrow><mml:mrow><mml:mn>2</mml:mn><mml:mi mathvariant="normal">+</mml:mi></mml:mrow></mml:msup></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi mathvariant="normal">i</mml:mi></mml:msub></mml:mrow></mml:math>[Ca2+]i levels in pigment epithelial (PE) cells, whereas depolarization evoked <mml:math><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msup><mml:mrow><mml:mtext>Ca</mml:mtext></mml:mrow><mml:mrow><mml:mn>2</mml:mn><mml:mi mathvariant="normal">+</mml:mi></mml:mrow></mml:msup></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi mathvariant="normal">i</mml:mi></mml:msub></mml:mrow></mml:math>[Ca2+]i elevations in both NPE and PE cells. Swelling-evoked <mml:math><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msup><mml:mrow><mml:mtext>Ca</mml:mtext></mml:mrow><mml:mrow><mml:mn>2</mml:mn><mml:mi mathvariant="normal">+</mml:mi></mml:mrow></mml:msup></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi mathvariant="normal">i</mml:mi></mml:msub></mml:mrow></mml:math>[Ca2+]i signals were inhibited by the TRPV4 antagonist HC067047 (IC50 ∼0.9 μM) and were absent in Trpv4−/− NPE. In NPE, but not PE, swelling-induced <mml:math><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msup><mml:mrow><mml:mtext>Ca</mml:mtext></mml:mrow><mml:mrow><mml:mn>2</mml:mn><mml:mi mathvariant="normal">+</mml:mi></mml:mrow></mml:msup></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi mathvariant="normal">i</mml:mi></mml:msub></mml:mrow></mml:math>[Ca2+]i signals required phospholipase A2 activation. TRPV4 localization to NPE was confirmed with immunolocalization and excitation mapping approaches, whereas in vivo MRI analysis confirmed TRPV4-mediated signals in the intact mouse ciliary body. Trpv2 and Trpv4 were the most abundant vanilloid transcripts in CB. Overall, our results support a model whereby TRPV4 differentially regulates cell volume, lipid, and calcium signals in NPE and PE cell types and therefore represents a potential target for antiglaucoma medications.