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X. Gasull, N. Comes, D. Soto, E. Ferrer, A. Gual; Mechanisms of Cell Volume Regulation in the Trabecular Meshwork: Modulation of Aqueous Humor Outflow . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3429.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Trabecular meshwork (TM) cell shape, volume, contractility and their interactions with extracellular matrix determine outflow facility. As we previously described, cell volume is essential to TM function (IOVS 38:2165-71, 1997). Here we further investigated the ionic channels and membrane receptors involved in regulatory volume decrease as well as their implications in outflow facility modulation. Methods: Primary cultures of TM cells from bovine eyes were used. K+ and Cl- currents were studied with whole-cell patch-clamp. Swelling was induced by an hypotonic shock. Intracellular Ca2+ concentration was measured in TM cells loaded with fura-2 using an epifluorescence microscope coupled to a CCD camera. Bovine anterior segments were perfused at constant pressure to measure outflow facility. Results: Patch-clamp recordings in hypotonic media activated both Cl- and K+ currents . Outward K+ currents were mediated by a BKCa channel previously described. Swelling-activated outwardly rectifying Cl- currents exhibited a time-dependent inactivation at high depolarizing potentials and rapidly reversed on return to isotonic conditions. Cl- currents were inhibited by a general Cl- channel blocker (DIDS, 100 µM) and a selective one (tamoxifen, 10 µM). Extracellular ATP (10 µM) activated Cl- currents in isotonic medium, but blocked them (1 mM) in hypotonic conditions. Since ATP-induced Cl- currents activation can be mediated via purinergic receptors, we examined their presence. We characterized a P2Y2 receptor with the following profile: ATP=UTP>ATP-γS>ADP=UDP. P2Y2 receptor activation triggers a Ca2+ release from intracellular stores via PLC activation. Hypotonic medium initially decreased outflow facility in perfused anterior segments, which recovered with time to baseline levels. Addition of tamoxifen (100 µM) lengthen the recovery phase, which implies Cl- currents participation in cell volume regulation. Conclusions: Cell swelling activates a regulatory volume decrease mechanism that implies activation of K+ and Cl- currents and participation of P2Y2 receptors. Since previous studies have shown that intracellular volume of TM cells is an important determinant of outflow facility, it seems feasible that cell volume regulation would be part of the homeostatic mechanisms of the TM and regulate the outflow pathway in response to changes in IOP.
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