Abstract
Purpose :
Micron-sized pores are routes for aqueous flow across an otherwise continuous inner wall endothelium of Schlemm’s canal. We have previously shown that stretch triggers pore formation in SC cells [1] through a calcium (Ca) dependent process [2]. We hypothesize that stretch opens mechanosensitive ion channels (MICs) such as TRPV4, allowing extracellular Ca [Ca]o influx that triggers Ca-induced Ca release (CICR) from internal stores. To test this hypothesis, we blocked MICs with gadolinium or with TRPV4 selective HC106. We also depleted Ca from the endoplasmic reticulum (ER) using cyclopiazonic acid (CPA) and varied [Ca]o.
Methods :
Human SC cells were isolated and characterised from two normal (SC73,SC75) and one glaucomatous (gSC90) donor [4]. Cells were seeded at low confluency onto a biotinylated gelatin-coated elastomeric membrane. Pre-stretch pores were labelled with green avidin then cells were treated with (A) Ca-free buffer (HBSS, 0.8mM Mg); (B) HBSS vehicle (1.3mM Ca) including 250mM gado, 20µM CPA or 1µM HC106; or (C) HBSS including 0.05µM, 0.2µM, 0.5µM, 1mM, 2mM or 5.5mM Ca. Cells were stretched to 48% areal increase for 3min and exposed to red avidin in Ca-free buffer for 1min to label transcellular pores formed during stretch. On average 220 cells were examined per case (156-358) by a masked observer, and pore number/cell compared using the E-test for Poisson statistics.
Results :
Ca-free buffer reduced pore formation relative to vehicle from 40 to 8 pores/100 cells in non-gSC cells and from 26 to 6 in gSC cells (p<0.001). Gado decreased pore formation relative to vehicle for all 3 cell strains (p<0.001), but not as effectively as zero Ca. In gSC cells, ER Ca depletion with CPA decreased pore formation (p<0.001) relative to vehicle, but TRPV4 block did not (p=0.06). In gSC cells there was a dose-dependent increase in pore count with increasing [Ca]o (Fig. B).
Conclusions :
Transcellular pore formation in SC cells requires [Ca]o entry through MICs followed by CICR from the ER. In gSC cells, blockade of TRPV4 did not inhibit pore formation, despite the effect of gado, suggesting that other MICs (e.g., piezo1) likely contribute to Ca entry.
[1] Braakman Exp Eye Res 2014 [2] Bertrand ARVO 2023 [3] Perkumas Exp Eye Res 2012.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.