Abstract
Purpose :
Intraocular pressure (IOP) is a mechanical oscillation with unknown physiological significance. TRPV4 (transient receptor potential isoform 4) channels partially mediate the trabecular meshwork (TM) response strain, shear, swelling and pressure. Their response to sustained stimulation is associated with stochastic regenerative [Ca2+]TM spikes. The goal was to elucidate the cellular mechanisms that underlie this process.
Methods :
Primary human TM cells were stimulated with the selective TRPV4 agonist GSK1016790A (GSK101). [Ca2+]i, [Na+]i and transmembrane currents measured with optical imaging and patch clamp in the presence/absence of pharmacological modulators. mRNA and protein levels were determined with qPCR, Western blot and spatial expression determined with antibody labeling. Cells were transfected with GFP probes, shRNAs and exposed to modulators of plasma membrane and intracellular Ca2+ signaling pathways.
Results :
Sustained TRPV4 activation triggered Ca2+ oscillations with periodicity of ~0.2 – 1 Hz, sustained depolarization and increase in [Na+]i. The oscillations were insensitive to blockers of Orai channels, voltage-operated Ca2+ and Na+ channels but could be mimicked with inhibition of Ca2+ sequestration into ER stores. Inhibition of mitochondrial Ca2+ signaling and suppression of TRPM4, a Ca2+-activated Na+ channel blocked the oscillations. However, pharmacological inhibition of TRPM4 had no effect on trabecular outflow and genetic ablation did not affect IOP.
Conclusions :
These results show that activation of pressure transducing ion channels in TM cells triggers amplitude- and time-dependent Ca2+ fluctuations that are regulated by steady-state [Ca2+]i and activation history. The fluctuations require continual interactions between multiple plasma membrane TRP channels, endoplasmic reticulum and mitochondrial calcium sequestration/release mechanisms. TM calcium homeostasis, therefore, is a sophisticated, multi-pathway process that time-dependently coordinates molecular activity of plasma membrane, cytosolic and store compartments.
This is a 2021 ARVO Annual Meeting abstract.