Activation of the BKCa Channel Increases Outflow Facility and Decreases Trabecular Meshwork Cell Volume

The trabecular meshwork (TM) of the eye plays a central role in modulating intraocular pressure by regulating aqueous humor outflow, although the mechanisms are largely unknown. We and others have shown previously that aqueous humor outflow facility is modulated by conditions that alter TM cell volume. We have also shown that the Na-K-Cl cotransport system is a primary regulator of TM cell volume and that its activity appears to be coordinated with net efflux pathways to maintain steady-state volume. However, the cellular mechanisms that regulate cotransport activity and cell volume in TM cells have yet to be elucidated. The present study was conducted to investigate the hypothesis that intracellular Cl concentration ([Cl]i) acts to regulate TM cell Na-K-Cl cotransport activity, as has been shown previously for some other cell types. We demonstrate here that the human TM cell Na-K-Cl cotransporter is highly sensitive to changes in [Cl]i. Our findings reveal a marked stimulation of Na-K-Cl cotransport activity, assessed as ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means of reducing [Cl]i. In contrast, preincubation of cells with media containing elevated K concentrations as a means of increasing [Cl]i results in inhibition of Na-K-Cl cotransport activity. The effects of reducing [Cl]i, as well as elevating [Cl]i, on Na-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced [Cl]i is additive with cell-shrinkage-induced stimulation of the cotransporter. Our studies also show that TM cell Na-K-Cl cotransport activity is altered by a variety of Cl channel modulators, presumably through changes in [Cl]i. These findings support the hypothesis that regulation of Na-K-Cl cotransport activity, and thus cell volume, by [Cl]i may participate in modulating outflow facility across the TM.

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Modulation of Aqueous Humor Outflow by Ionic Mechanisms Involved in Trabecular Meshwork Cell Volume Regulation

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.04-0060 ◽

2004 ◽

Vol 45 (10) ◽

pp. 3650 ◽

Cited By ~ 37

Author(s):

David Soto ◽

Nu´ria Comes ◽

Elisa Ferrer ◽

Miguel Morales ◽

Artur Escalada ◽

...

Keyword(s):

Cell Volume ◽

Aqueous Humor ◽

Trabecular Meshwork ◽

Volume Regulation ◽

Cell Volume Regulation ◽

Trabecular Meshwork Cell ◽

Aqueous Humor Outflow ◽

Ionic Mechanisms

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NO-induced regulation of human trabecular meshwork cell volume and aqueous humor outflow facility involve the BKCa ion channel

AJP Cell Physiology ◽

10.1152/ajpcell.00363.2007 ◽

2008 ◽

Vol 294 (6) ◽

pp. C1378-C1386 ◽

Cited By ~ 56

Author(s):

William M. Dismuke ◽

Chigozirim C. Mbadugha ◽

Dorette Z. Ellis

Keyword(s):

Cell Volume ◽

Trabecular Meshwork ◽

Time Course ◽

Soluble Guanylate Cyclase ◽

Anterior Segment ◽

Drug Application ◽

Cellular Mechanisms ◽

No Donors ◽

Outflow Facility ◽

Aqueous Humor Outflow

Nitric oxide (NO) donors decrease intraocular pressure (IOP) by increasing aqueous outflow facility in the trabecular meshwork (TM) and/or Schlemm's canal. However, the cellular mechanisms are unknown. Cellular mechanisms known to regulate outflow facility include changes in cell volume and cellular contractility. In this study, we investigated the effects of NO donors on outflow facility and NO-induced effects on TM cell volume. We tested the involvement of soluble guanylate cyclase (sGC), cGMP, PKG, and the large-conductance Ca2+-activated K+ (BKCa) channel using inhibitors and activators. Cell volume was measured using calcein AM fluorescent dye, detected by confocal microscopy, and quantified using NIH ImageJ software. An anterior segment organ perfusion system measured outflow facility. NO increased outflow facility in porcine eye anterior segments (0.4884–1.3956 μl·min−1·mmHg−1) over baseline (0.2373–0.5220 μl·min−1·mmHg−1) within 10 min of drug application. These NO-induced increases in outflow facility were inhibited by the the BKCa channel inhibitor IBTX. Exposure of TM cells to NO resulted in a 10% decrease in cell volume, and these decreases were abolished by the sGC inhibitor 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and IBTX, suggesting the involvement of sGC and K+ eflux, respectively. NO-induced decreases in cell volume were mimicked by 8-Br-cGMP and abolished by the PKG inhibitor (RP)-8-Br-PET-cGMP-S, suggesting the involvement cGMP and PKG. Additionally, the time course for NO-induced decreases in TM cell volume correlated with NO-induced increases in outflow facility, suggesting that the NO-induced alterations in cell volume may influence outflow facility.

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Circumferential trabecular meshwork cell density in the human eye

Experimental Eye Research ◽

10.1016/j.exer.2021.108494 ◽

2021 ◽

Vol 205 ◽

pp. 108494

Author(s):

Markus H. Kuehn ◽

Janice A. Vranka ◽

David Wadkins ◽

Thomas Jackson ◽

Lin Cheng ◽

...

Keyword(s):

Cell Density ◽

Trabecular Meshwork ◽

Human Eye ◽

Trabecular Meshwork Cell

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Cyclic Mechanical Stress and Trabecular Meshwork Cell Contractility

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.08-2694 ◽

2009 ◽

Vol 50 (8) ◽

pp. 3826 ◽

Cited By ~ 23

Author(s):

Renata F. Ramos ◽

Grant M. Sumida ◽

W. Daniel Stamer

Keyword(s):

Mechanical Stress ◽

Trabecular Meshwork ◽

Cell Contractility ◽

Trabecular Meshwork Cell

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Human trabecular meshwork cell volume regulation

AJP Cell Physiology ◽

10.1152/ajpcell.00544.2001 ◽

2002 ◽

Vol 283 (1) ◽

pp. C315-C326 ◽

Cited By ~ 41

Author(s):

Claire H. Mitchell ◽

Johannes C. Fleischhauer ◽

W. Daniel Stamer ◽

K. Peterson-Yantorno ◽

Mortimer M. Civan

Keyword(s):

Cell Volume ◽

Trabecular Meshwork ◽

Regulatory Volume Decrease ◽

Cell Volume Regulation ◽

Channel Blockers ◽

Uptake Mechanism ◽

Fluid Uptake ◽

Intracellular Ca ◽

Predominant Effect ◽

Volume Decrease

The volume of certain subpopulations of trabecular meshwork (TM) cells may modify outflow resistance of aqueous humor, thereby altering intraocular pressure. This study examines the contribution that Na+/H+, Cl−/HCO[Formula: see text]exchange, and K+-Cl− efflux mechanisms have on the volume of TM cells. Volume, Cl− currents, and intracellular Ca2+ activity of cultured human TM cells were studied with calcein fluorescence, whole cell patch clamping, and fura 2 fluorescence, respectively. At physiological bicarbonate concentration, the selective Na+/H+ antiport inhibitor dimethylamiloride reduced isotonic cell volume. Hypotonicity triggered a regulatory volume decrease (RVD), which could be inhibited by the Cl− channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), the K+channel blockers Ba2+ and tetraethylammonium, and the K+-Cl− symport blocker [(dihydroindenyl)oxy]alkanoic acid. The fluid uptake mechanism in isotonic conditions was dependent on bicarbonate; at physiological levels, the Na+/H+ exchange inhibitor dimethylamiloride reduced cell volume, whereas at low levels the Na+-K+-2Cl− symport inhibitor bumetanide had the predominant effect. Patch-clamp measurements showed that hypotonicity activated an outwardly rectifying, NPPB-sensitive Cl− channel displaying the permeability ranking Cl− > methylsulfonate > aspartate. 2,3-Butanedione 2-monoxime antagonized actomyosin activity and both increased baseline [Ca2+] and abolished swelling-activated increase in [Ca2+], but it did not affect RVD. Results indicate that human TM cells display a Ca2+-independent RVD and that volume is regulated by swelling-activated K+ and Cl− channels, Na+/H+ antiports, and possibly K+-Cl− symports in addition to Na+-K+-2Cl− symports.

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