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

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) TRPV4 | ciliary body | intraocular pressure | aqueous humor | glaucoma F ormation of aqueous humor in the vertebrate eye takes place within the ciliary body (CB), a highly folded tissue consisting of pigmented epithelial (PE) cells, nonpigmented epithelial (NPE) cells, and the ciliary muscle (1, 2). Together, PE cells, which face the vascularized stroma and represent a forward continuation of the retinal pigment epithelium (RPE), and NPE cells, which face the posterior chamber (lumen) of the eye and extend the neuronal retina, form the blood-aqueous barrier and regulate the production and secretion of aqueous humor. The aqueous fluid supplies nutrients and oxygen to nonvascularized tissues (lens, cornea, and trabecular meshwork) and is ultimately drained through the ciliary muscle and the trabecular meshwork in the anterior chamber of the eye. Aqueous secretion is subserved by the unidirectional transport of ions and water through gap junctions between PE cells and NPE cells (3,4) and is driven by the osmotic gradient generated by Na + /K + exchange across basolateral NPE membranes (2-5). Despite the critical dependence of aqueous humor secretion on osmotic pressure (1, 4, 6), the molecular mechanism through which NPE and PE cells sense and regulate changes in volume is not well understood.In addition to osmotic shifts, CB cells experience mechanical forces associated with mean and time-varying aspects of intraocular pressure (IOP), a phenomenon that reflects balanced regulation of fluid secretion from NPE cells and its drainage from the anterior eye. Excessive IOP elevations represent the primary, and major, risk factor for contracting glaucoma (6, 7), an optic neuropathy that represents the second leading cause of blindness in the world. Therefore, aqueous secretion is often targeted by antiglaucoma medications that include β-adrenergic receptor antagonists, carbonic anhydrase inhibitors, α 2 -adrenergic agonists, and muscarinic cholinergic agonists (7). A key question, however, is whether CB cells themselves are able to sense force mediated by membrane stretch induced by hydrostatic pressure or swelling, and what such mechanisms might be.Here, we identify a key osmosensor in CB as transient receptor potential channel vanilloid isoform 4 (TRPV4), a polymodal nonselective cation-permeable channel that has been implicated in mechanotransduction (8, 9) as well as regulation of paracellular permeability in multiple epithelial tissues (10-15). Intriguingly, we found that TRPV4 is selectively distributed across CB by being confined to the NPE and excluded from PE cells. We characterized the functional role of TRPV4 as the predominant NPE swelling sensor and determined its contribution to...

Interactions of the Mechanosensitive Channels with Extracellular Matrix, Integrins, and Cytoskeletal Network in Osmosensation

“…(2) Prolonged hyperosmotic stress promotes VP gene transcription and translation and RVI (Figure 1Bc) while maintaining ECM-integrin-associated gate opening. (3) Early hyposmotic tension causes mild conformational change in integrins-TRP complex, inhibition of TRPV channels by their uncoupling with microtubule network and the “leakage” of these channels (Muraki et al, 2003; Sato et al, 2013; Jo et al, 2016) including ATP. ATP activation of purine-associated opening of TRPC and other TRP channels (Goel et al, 2007; Soya et al, 2014) can quickly reverse this initial inhibition and lead to cytosolic Ca 2+ increase.…”

“…Further studies reveal that these SICs could also be stretch-activated cation channels because hyposmotic challenges can increase intracellular Ca 2+ concentration through activation of TRPV1, TRPV2, TRPV4 in Merkel cells from hamster buccal mucosa (Soya et al, 2014), TRPV4 in acinar cells (Aure et al, 2010) and in nonpigmented epithelial cells (Jo et al, 2016). Consistently, in acute hyponatremic condition, serum VP levels increase significantly following initial inhibition (Yagil and Sladek, 1990), which reflects a reactivation of VP neurons following the initial inhibition (Wang et al, 2013a,b) through the mechanism of “resetting osmosensory threshold at the local neural circuit” (Wang et al, 2011).…”

“…Thus, an increased interactions between microtubule network with TRPV1 during cell shrinkage could account for hyperosmotic activation of osmosensory neurons (Prager-Khoutorsky and Bourque, 2015). However, this hypothesis could not explain hyposmotic intracellular Ca 2+ increase (Aure et al, 2010; Soya et al, 2014; Jo et al, 2016), the recovery of VP neuronal activity from hyposmotic inhibition (Wang et al, 2013a,b) and the increased VP secretion during volemic increase in chronic osmotic stress (Zhang et al, 2001). Here, referring to the hearing mechanism (Sukharev and Corey, 2004; Martinac, 2014), we propose that if hyperosmotic activation of TRP channels is due to a “push” of microtubule network (Prager-Khoutorsky and Bourque, 2015), the hyposmotic activation of TRP channels should be because of a “pull” of the network in coordination with conformational changes in other cellular components (Figure 1A).…”

Subcellular propagation of calcium waves in Müller glia does not require autocrine/paracrine purinergic signaling

“…17,18,48 Acutely dissociated retinal M€ uller cells were loaded with the ratiometric fluorescent dye fura-2 AM (Life Sciences) for 30 min. To allow complete de-esterification of intracellular AM esters, cells were incubated in dye-free L-15 medium for 30 min.…”

TRPV4 Does Not Regulate the Distal Retinal Light Response

“…Its prominent expression across auditory, nociceptive, thermosensitive, mechanosensitive, and visual pathways (White et al 2016; Redmon et al 2017) is suggestive of important functional roles in sensory signaling. The strong expression levels of TRPV4 mRNA/protein in the cornea (Mergler et al 2012), ciliary body (Jo et al 2016), trabecular meshwork (Ryskamp et al 2016), lens (Shahidullah et al 2012), and retina (Ryskamp et al 2011) predict that this polymodal nonselective cation channel contributes to the regulation of aqueous fluid transport, accommodation, ocular pressure sensing, endocannabinoid signaling, and/or volume regulation in the eye (Krizaj et al 2014; Krizaj 2016); however whether and what roles TRPV4 plays in visual signaling is unknown. Here, we show that TRPV4 activation in Müller cells, radial glia that play a key role in retinal volume regulation and transretinal propagation of calcium waves (Jo et al 2015; Phuong et al 2016), induces a calcium response in adjacent photoreceptors.…”