Regulatory Roles of Anoctamin-6 in Human Trabecular Meshwork Cells.

Purpose: Trabecular meshwork (TM) cell volume is a determinant of aqueous humor outflow resistance, and thereby IOP. Regulation of TM cell volume depends on chloride ion (Cl-) release through swelling-activated channels (ICl,Swell), whose pore is formed by LRRC8 proteins. Chloride ion release through swelling-activated channels has been reported to be regulated by calcium-activated anoctamins, but this finding is controversial.

Effects of cardiotonic steroids on trabecular meshwork cells: search for mediator of ouabain-enhanced outflow facility.

Lowering intraocular pressure (IOP) is currently the only strategy documented to slow the onset and progression of glaucomatous blindness. Ouabain, a cardiotonic glycoside inhibitor of Na(+), K(+)-activated ATPase, was recently reported to enhance outflow facility in porcine anterior segments at concentrations as low as 30 nM for ≥4 h, suggesting a novel approach to lowering IOP. The underlying mechanism is unknown, but associated cytoskeletal changes were observed in porcine trabecular meshwork cells.

Mechanisms of ATP release, the enabling step in purinergic dynamics.

The only effective intervention to slow onset and progression of glaucomatous blindness is to lower intraocular pressure (IOP). Among other modulators, adenosine receptors (ARs) exert complex regulation of IOP. Agonists of A(3)ARs in the ciliary epithelium activate Cl(-) channels, favoring increased formation of aqueous humor and elevated IOP.

Cytoskeletal dependence of adenosine triphosphate release by human trabecular meshwork cells.

Purpose: To test whether adenosine triphosphate (ATP) release links cytoskeletal remodeling with release of matrix metalloproteinases (MMPs), regulators of outflow facility and intraocular pressure.

Methods: ATP release was measured by luciferin-luciferase. Ecto-ATPases from transformed human trabecular meshwork (TM) cells (TM5) and explant-derived TM cells were identified by RT-PCR.

Mechanisms of ATP release by human trabecular meshwork cells, the enabling step in purinergic regulation of aqueous humor outflow.

Our guiding hypothesis is that ecto-enzymatic conversion of extracellular ATP to adenosine activates A(1) adenosine receptors, reducing resistance to aqueous humor outflow and intraocular pressure. The initial step in this purinergic regulation is ATP release from outflow-pathway cells by mechanisms unknown. We measured similar ATP release from human explant-derived primary trabecular meshwork (TM) cells (HTM) and a human TM cell line (TM5).

Pathways for ATP release by bovine ciliary epithelial cells, the initial step in purinergic regulation of aqueous humor inflow.

ATP release by nonpigmented (NPE) and pigmented (PE) ciliary epithelial cells is the enabling step in purinergic regulation of aqueous humor formation, but the release pathways are unknown. We measured ATP release from primary cultures of bovine mixed NPE and PE (bCE) cells and transformed bovine NPE and PE cells, using the luciferin-luciferase reaction. Hypotonicity-triggered bCE ATP release was inhibited by the relatively selective blocker of pannexin-1 (PX1) hemichannels (probenecid, 1 mM, 47 ± 2%), by a connexin inhibitor (heptanol, 1 mM, 49 ± 4%), and by an inhibitor of vesicular release (bafilomycin A1, 25 ± 2%), but not by the P2X(7) receptor (P2RX(7)) antagonist KN-62.

Nucleoside-derived antagonists to A3 adenosine receptors lower mouse intraocular pressure and act across species.

The purpose of the study was to determine whether novel, selective antagonists of human A3 adenosine receptors (ARs) derived from the A3-selective agonist Cl-IB-MECA lower intraocular pressure (IOP) and act across species. IOP was measured invasively with a micropipette by the Servo-Null Micropipette System (SNMS) and by non-invasive pneumotonometry during topical drug application. Antagonist efficacy was also assayed by measuring inhibition of adenosine-triggered shrinkage of native bovine nonpigmented ciliary epithelial (NPE) cells.

Cell-specific differential modulation of human trabecular meshwork cells by selective adenosine receptor agonists.

Activation of A1 and A2A subtype adenosine receptors (AR) likely exert opposing effects on outflow of aqueous humor, and thereby, on intraocular pressure. Selective agonists of adenosine receptor (AR) subtypes have previously been applied to trabecular meshwork (TM) and Schlemm's canal (SC) cells to identify the site(s) of differential purinergic modulation. However, the apparent changes in volume monitored by previously measuring projected cell area might have partially reflected cell contraction and relaxation.

Swelling-activated Cl- channels support Cl- secretion by bovine ciliary epithelium.

Purpose: To determine whether swelling-activated Cl- currents (ICl,swell) observed in isolated nonpigmented ciliary epithelial (NPE) cells contribute to Cl- secretion across the ciliary epithelium.

Methods: Ion transport across intact bovine ciliary epithelium was monitored electrically. Native isolated bovine NPE cells were harvested enzymatically.

The cross-species A3 adenosine-receptor antagonist MRS 1292 inhibits adenosine-triggered human nonpigmented ciliary epithelial cell fluid release and reduces mouse intraocular pressure.

Purpose: Antagonists to A3 adenosine receptors (ARs) lower mouse intraocular pressure (IOP), but extension to humans is limited by species variability. We tested whether the specific A3AR antagonist MRS 1292, designed to cross species, mimicks the effects of other A3AR antagonists on cultured human nonpigmented ciliary epithelial (NPE) cells and mouse IOP.

Methods: NPE cell volume was monitored by electronic cell sorting.

Differential P1-purinergic modulation of human Schlemm's canal inner-wall cells.

Intraocular pressure is directly dependent on aqueous humor flow into, and resistance to flow out of, the eye. Adenosine has complex effects on intraocular pressure. Stimulation of A1 and A2A adenosine receptors changes intraocular pressure oppositely, likely through opposing actions on the outflow of aqueous humor.

cAMP-activated maxi-Cl(-) channels in native bovine pigmented ciliary epithelial cells.

The eye's aqueous humor is secreted by a bilayered ciliary epithelium comprising pigmented (PE) and nonpigmented (NPE) epithelial cell layers. Stromal Cl(-) enters the PE cells and crosses gap junctions to the NPE cells for release into the aqueous humor. Maxi-Cl(-) channels are expressed in PE cells, but their physiological significance is unclear.

Common actions of adenosine receptor agonists in modulating human trabecular meshwork cell transport.

A(1) adenosine receptors (ARs) reduce, and A(2)ARs increase intraocular pressure, partly by differentially altering resistance to aqueous humor outflow. It is unknown whether the opposing effects of A(1)AR and A(2)AR agonists are mediated at different outflow-pathway cell targets or by opposing actions on a single cell target. We tested whether a major outflow-pathway cell, the trabecular meshwork (TM) cell might constitute the primary AR-agonist target and respond differentially to A(1), A(2A) and A(3)AR agonists.

Human trabecular meshwork cell volume regulation.

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 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.

PGE(2), Ca(2+), and cAMP mediate ATP activation of Cl(-) channels in pigmented ciliary epithelial cells.

Purines regulate intraocular pressure. Adenosine activates Cl(-) channels of nonpigmented ciliary epithelial cells facing the aqueous humor, enhancing secretion. Tamoxifen and ATP synergistically activate Cl(-) channels of pigmented ciliary epithelial (PE) cells facing the stroma, potentially reducing net secretion.

Timolol may inhibit aqueous humor secretion by cAMP-independent action on ciliary epithelial cells.

The beta-adrenergic antagonist timolol reduces ciliary epithelial secretion in glaucomatous patients. Whether inhibition is mediated by reducing cAMP is unknown. Elemental composition of rabbit ciliary epithelium was studied by electron probe X-ray microanalysis.

Na+/H+ and CI-/HCO3-antiporters of bovine pigmented ciliary epithelial cells.

Medical therapy of glaucoma commonly aims at slowing aqueous humor formation by the ocular ciliary epithelial bilayer, but underlying mechanisms are poorly understood. The first step in secretion is NaCl uptake from the stroma into the pigmented ciliary epithelial (PE) cell layer by electroneutral transporters. After crossing gap junctions into the nonpigmented ciliary epithelial (NPE) cell layer, solute is released into the aqueous humor.

Similarity of A(3)-adenosine and swelling-activated Cl(-) channels in nonpigmented ciliary epithelial cells.

Chloride release from nonpigmented ciliary epithelial (NPE) cells is a final step in forming aqueous humor, and adenosine stimulates Cl(-) transport by these cells. Whole cell patch clamping of cultured human NPE cells indicated that the A(3)-selective agonist 1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl-be ta-D-ribofuranuronamide (IB-MECA) stimulated currents (I(IB-MECA)) by approximately 90% at +80 mV. Partial replacement of external Cl(-) with aspartate reduced outward currents and shifted the reversal potential (V(rev)) from -23 +/- 2 mV to -0.

Tamoxifen and ATP synergistically activate Cl- release by cultured bovine pigmented ciliary epithelial cells.

Purines alter aqueous humour secretion by the bilayered ciliary epithelium. Adenosine but not ATP shrinks non-pigmented ciliary epithelial (NPE) cells by activating Cl- channels. We now report effects of ATP on pigmented ciliary epithelial (PE) cells.

A3 adenosine receptors regulate Cl- channels of nonpigmented ciliary epithelial cells.

Adenosine stimulates Cl- channels of the nonpigmented (NPE) cells of the ciliary epithelium. We sought to identify the specific adenosine receptors mediating this action. Cl- channel activity in immortalized human (HCE) NPE cells was determined by monitoring cell volume in isotonic suspensions with the cationic ionophore gramicidin present.

Potential contribution of epithelial Na+ channel to net secretion of aqueous humor.

The aqueous humor of the eye is secreted by the bilayered ciliary epithelium, consisting of the pigmented (PE) cell layer facing the stroma and the nonpigmented (NPE) cell layer facing the aqueous humor. Cells within each layer and between the two layers are linked by gap junctions, forming a ciliary epithelial syncytium. Unidirectional secretion from the stroma to the aqueous proceeds both through the cells (the transcellular pathway) and between the cells (the paracellular pathway).

Adenosine stimulates Cl- channels of nonpigmented ciliary epithelial cells.

Ciliary epithelial cells possess multiple purinergic receptors, and occupancy of A1 and A2 adenosine receptors is associated with opposing effects on intraocular pressure. Aqueous adenosine produced increases in short-circuit current across rabbit ciliary epithelium, blocked by removing Cl- and enhanced by aqueous Ba2+. Adenosine's actions were further studied with nonpigmented ciliary epithelial (NPE) cells from continuous human HCE and ODM lines and freshly dissected bovine cells.

Effect of heptanol on the short circuit currents of cornea and ciliary body demonstrates rate limiting role of heterocellular gap junctions in active ciliary body transport.

Rabbit ciliary body and cornea were mounted in Ussing-type chambers in Tyrode's under voltage clamp and the effects of heptanol, a gap junction inhibitor, on the short circuit current generated by each of the respective epithelia were determined. Studies were carried out either in control conditions or following amphotericin B permeabilization of either the basolateral membrane of the nonpigmented epithelium of the ciliary body or the apical membrane of the corneal epithelium, respectively. Previous studies have shown that, following these permeabilizations, short circuit currents are established, reflecting aqueous (or tear)-to-serosa Na+ fluxes, and that Na+ translocation through gap junctions connecting the individual layers of these tissues constitutes the major rate limiting step.

Regulation of epithelial Na+ channels from M-1 cortical collecting duct cells.

The M-1 cell line is derived from the mouse cortical collecting duct and displays the low-conductance, highly Na(+)-selective channel activity of the alpha,beta, gamma-heterotrimeric epithelial Na+ channel (ENaC). The short-circuit current (Isc) across M-1 monolayers was 89 +/- 4 microA/cm2, and the transepithelial conductance was 2.1 +/- 0.

Regulation of epithelial Na+ permeability by protein kinase C is tissue specific.

Protein kinase C (PKC) is a major regulator of a broad range of cellular functions. Activation of PKC has been reported to stimulate Na+ transport across frog skin epithelium by increasing the apical Na+ permeability. This positive natriferic response has not been observed with other epithelial preparations, and could reflect the specific experimental conditions of different laboratories, or species or organ specificity of the response to PKC.