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. Particularly uncertain has been the effect of anoctamin Ano6, reported as a Ca2+-activated Cl- (CaCC) or cation channel in other cells. The current study tested whether anoctamin activity modifies volume regulation of primary TM cell cultures and cell lines. Methods: Gene expression was studied with quantitative PCR, supplemented by reverse-transcriptase PCR and Western immunoblots. Currents were measured by ruptured whole-cell patch clamping and volume by electronic cell sizing. Results: Primary TM cell cultures and the TM5 and GTM3 cell lines expressed Ano6 3 to 4 orders of magnitude higher than the other anoctamin CaCCs (Ano1 and Ano2). Ionomycin increased cell Ca2+ and activated macroscopic currents conforming to CaCCs in other cells, but displayed significantly more positive mean reversal potentials (+5 to +12 mV) than those displayed by ICl,Swell (-14 to -21 mV) in the same cells. Nonselective CaCC inhibitors (tannic acid>CaCCinh-A01) and transient Ano6 knockdown strongly inhibited ionomycin-activated currents, ICl,Swell and the regulatory volume response to hyposmotic swelling. Conclusions: Ionomycin activates CaCCs associated with net cation movement in TM cells. These currents, ICl,Swell, and cell volume are regulated by Ano6. The findings suggest a novel clinically-relevant approach for altering cell volume, and thereby outflow resistance, by targeting Ano6.

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. We have previously found that changes in ATP release and subsequent ectoenzymatic conversion to adenosine may play a role in linking cytoskeletal remodeling with modulation of outflow resistance. We now tested whether altered ATP release might also be a mediator of ouabain's effect on outflow facility. ATP release from transformed human TM5 and explant-derived human trabecular meshwork cells was measured by the luciferin-luciferase reaction. Matrix metalloproteinases (MMPs) were studied by zymography, cell Na(+) concentration by SBFI fluorometry, gene expression of ATP-release pathways by real-time PCR, cell volume by electronic cell sorting and cell viability by the LDH and MTT methods. Actin was examined by confocal microscopy of phalloidin-stained cells. Contrary to expectation, ouabain at concentrations ≥10 nM inhibited swelling-triggered ATP release from TM5 cells after ≥4 h of exposure. Inhibition was enhanced by increasing ouabain concentration and exposure time. Similar effects were produced by the reversible cardiac aglycone strophanthidin. Ouabain also inhibited swelling-activated ATP release from explant-derived native human TM cells. Ouabain (4 h, 30 nM and 100 nM) did not alter gene expression of the ATP-release pathways, and cell viability was unchanged by exposure to ouabain (30 nM-1 µM). Preincubation with 30 nM ouabain for 4 h did not detectably change Na(+) level, the regulatory volume decrease (RVD) or the actin cytoskeleton of TM5 cells, but did inhibit hypotonicity-elicited ATP release. Moreover, even when N-methyl-d-glucosamine replaced Na(+) in the extracellular fluid, ouabain still inhibited swelling-initiated ATP release at 100 nM. In the absence of ouabain, extracellular ATP stimulated MMP secretion, which was largely blocked by inhibiting conversion of ATP to adenosine, as expected. In contrast, ouabain reduced ATP release, but did not alter secretion of MMP-2 and MMP-9 from cells pretreated for ≤4 h. The results suggest that: (1) ouabain can trigger enhancement of outflow facility independent of its transport and actin-restructuring effects exerted at higher concentration and longer duration; (2) ouabain exerts parallel independent effects on ATP release and outflow facility; and (3) these effects likely reflect ouabain-induced changes in the scaffolding and/or signaling functions of Na(+), K(+)-activated ATPase.

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). Responses to 21 inhibitors indicated that pannexin-1 (PX1) and connexin (Cx) hemichannels and P2X(7) receptors (P2RX(7) ) were comparably important in modulating ATP release induced by hypotonic swelling, whereas vesicular release was insignificant. Consistent with prior studies of PX1 activity in certain other cells, ATP release was lowered by the reducing agent dithiothreitol. Overexpressing PX1 in HEK293T cells promoted, while partial knockdown (KD) in both HEK293T and TM5 cells inhibited hypotonicity-activated ATP release. Additionally, KD reduced the pharmacologically defined contribution of PX1 and enhanced those of Cx and P2RX(7) . ATP release was also triggered by raising intracellular Ca(2+) activity with ionomycin after a prolonged lag time and was unaffected by the PX1 blocker probenecid, but nearly abolished by P2RX(7) antagonists. We conclude that swelling-stimulated ATP release from human TM cells is physiologically mediated by PX1 and Cx hemichannels and P2X(7) receptors, but not by vesicular release. PX1 appears not to be stimulated by intracellular Ca(2+) in TM cells, but can be modulated by oxidation-reduction state. The P2RX(7) -dependent component of swelling-activated release may be mediated by PX1 hemichannels or reflect apoptotic magnification of ATP release, either through itself and/or hemichannels.

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. In contrast, stimulating A(1)ARs in the trabecular outflow pathway enhances release of matrix metalloproteinases (MMPs) from trabecular meshwork (TM) cells, reducing resistance to outflow of aqueous humor to lower IOP. These opposing actions are thought to be initiated by cellular release of ATP and its ectoenzymatic conversion to adenosine. This view is now supported by our identification of six ectoATPases in trabecular meshwork (TM) cells and by our observation that external ATP enhances TM-cell secretion of MMPs through ectoenzymatic formation of adenosine. ATP release is enhanced by cell swelling and stretch. Also, enhanced ATP release and downstream MMP secretion is one mediator of the action of actin depolymerization to reduce outflow resistance. Inflow and outflow cells share pannexin-1 and connexin hemichannel pathways for ATP release. However, vesicular release and P2X(7) release pathways were functionally limited to inflow and outflow cells, respectively, suggesting that blocking exocytosis might selectively inhibit inflow, lowering 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. Actin was visualized by phalloidin staining. Cell viability was assayed by lactate dehydrogenase and thiazolyl blue tetrazolium bromide methods and propidium iodide exclusion, gene expression by real-time PCR, and MMP release by zymography. Cell volume was monitored by electronic cell sorting. RESULTS: Hypotonicity (50%) and mechanical stretch increased ATP release with similar pharmacologic profiles. TM cells expressed ecto-ATPases E-NPP1-3, E-NTPD2, E-NTPD8, and CD73. Prolonged dexamethasone (DEX) exposure (≥ 2 weeks), but not brief exposure (3 days), increased cross-linked actin networks and reduced swelling-triggered ATP release. Cytochalasin D (CCD) exerted opposite effects. Neither DEX nor CCD altered the cell viability, gene expression, or pharmacologic profile of ATP-release pathways. DEX accelerated, and CCD slowed, the regulatory volume decrease after hypotonic exposure. Activating A(1) adenosine receptors (A(1)ARs) increased total MMP-2 and MMP-9 release. DEX reduced total A(1)AR-triggered MMP release, and CCD increased the active form of MMP-2 release. The A(1)AR agonist CHA and the A(1)AR antagonist DPCPX partially reversed the effects of DEX and CCD, respectively. CONCLUSIONS: Cytoskeletal restructuring modulated swelling-activated ATP release, in part by changing the duration of cell swelling after hypotonic challenge. Modifying ATP release is expected to modulate MMP secretion by altering ecto-enzymatic delivery of adenosine to A(1)ARs, linking cytoskeletal remodeling and MMP-mediated modulation of outflow facility.

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. Bafilomycin A1 acts by reducing the driving force for uptake of ATP from the cytosol into vesicles. The reducing agent dithiothreitol reduced probenecid-blockable ATP release. Similar results were obtained with NPE and PE cell lines. Pannexins PX1-3, connexins Cx43 and Cx40, and P2RX(7) were identified in native cells and cell lines by RT-PCR. PX1 mRNA expression was confirmed by Northern blots; its quantitative expression was comparable to that of Cx43 by real-time PCR. Heterologous expression of bovine PX1 in HEK293T cells enhanced swelling-activated ATP release, inhibitable by probenecid. We conclude that P2RX(7)-independent PX1 hemichannels, Cx hemichannels, and vesicular release contribute comparably to swelling-triggered ATP release. The relatively large response to dithiothreitol raises the possibility that the oxidation-reduction state is a substantial regulator of PX1-mediated ATP release from bovine ciliary epithelial cells.

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. Five agonist-based A3AR antagonists lowered mouse IOP measured with SNMS tonometry by 3-5 mm Hg within minutes of topical application. Of the five agonist derivatives, LJ 1251 was the only antagonist to lower IOP measured by pneumotonometry. No effect was detected pneumotonometrically over 30 min following application of the other four compounds, consonant with slower, smaller responses previously measured non-invasively following topical application of A3AR agonists and the dihydropyridine A3AR antagonist MRS 1191. Latanoprost similarly lowered SNMS-measured IOP, but not IOP measured non-invasively over 30 min. Like MRS 1191, agonist-based A3AR antagonists applied to native bovine NPE cells inhibited adenosine-triggered shrinkage. In summary, the results indicate that antagonists of human A3ARs derived from the potent, selective A3 agonist Cl-IB-MECA display efficacy in mouse and bovine cells, as well. When intraocular delivery was enhanced by measuring mouse IOP invasively, five derivatives of the A3AR agonist Cl-IB-MECA lowered IOP but only one rapidly reduced IOP measured non-invasively after topical application. We conclude that derivatives of the highly-selective A3AR agonist Cl-IB-MECA can reduce IOP upon reaching their intraocular target, and that nucleoside-based derivatives are promising A3 antagonists for study in multiple animal models.

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. In addition, whole-cell current responses of the TM cells previously described were highly variable following application of selective A1, A2A and A3 agonists. The complexity of the electrophysiologic responses may have reflected cell heterogeneity of the populations harvested from collagenase digestion of TM explants. We now report measurements of TM-cell volume using calcein fluorescence quenching, an approach independent of contractile state. Furthermore, we have applied selective AR agonists to a uniform population of human TM cells, the hTM5 cell line. A1, but not A2A or A3, AR agonists triggered TM-cell shrinkage. Both A1 and A2A AR agonists produced reproducible increases in TM-cell whole-cell currents of similar magnitude. The results suggest that previous measurements of explant-derived TM cells may have reflected a range of responses from phenotypically different cell populations, and that the opposing effects of A1 and A2A agonists on outflow resistance are not likely to be mediated by actions on a single population of TM cells. These opposing effects might reflect AR responses by two or more subpopulations of TM cells, by TM and SC cells or by inner-wall SC cells, alone.

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. Cell volume changes were measured by calcein-fluorescence quenching. RESULTS: Bilateral reduction in osmolality transiently increased short-circuit current (Isc), averaging 60% to 70%. Bilateral pretreatment with 5-nitro-2-(phenylpropylamino)-benzoate (NPPB), a Cl- channel blocker, reduced Isc stimulation by approximately 60%, suggesting that transcellular ICl,swell largely mediates the increased current. The hypotonically-triggered Isc stimulation was also inhibited by phloretin, a blocker of swelling-activated Cl- channels and by flufenamic acid, a blocker of Cl- and nonselective cation channels. Cyclamate substitution for bath Cl- reduced the baseline Isc and the increase in hypotonically-triggered Isc. In that case, addition of either NPPB or flufenamic acid did not produce further inhibition. The transepithelial responses were correlated with regulatory volume responses of freshly harvested NPE cells. Hypotonicity elicited a regulatory volume decrease (RVD) over a period comparable to that of the hypotonicity-triggered increase in Isc. The RVD was also inhibited by Cl--channel blockers and by Cl- substitution. CONCLUSIONS: ICl,swell of NPE cells is functionally expressed in intact ciliary epithelium and is oriented to subserve aqueous humor formation. NPE cell volume can be measured with calcein-fluorescence quenching. ICl,swell may be stimulated by increased stromal fluid uptake and delivery to the NPE cells, facilitating Cl- secretion and increasing fluid release into the posterior chamber.

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. Mouse IOP was measured with the Servo-Null Micropipette System. RESULTS: Adenosine triggered A3AR-mediated shrinkage of human NPE cells. Shrinkage was blocked by MRS 1292 (IC50 = 42 +/- 11 nM, p < 0.01) and by another A3AR antagonist effective in this system, MRS 1191. Topical application of the A3AR agonist IB-MECA increased mouse IOP. MRS 1292 reduced IOP by 4.0 +/- 0.8 mmHg at 25-microM droplet concentration (n = 10, p < 0.005). CONCLUSIONS: MRS 1292 inhibits A3AR-mediated shrinkage of human NPE cells and reduces mouse IOP, consistent with its putative action as a cross-species A3 antagonist.

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. While the cellular sites regulating outflow resistance are unknown, the cells lining the inner wall of Schlemm's canal (SC) are a likely regulatory site. We applied selective adenosine receptor agonists to SC cells in vitro to compare the responses to A1 and A2A stimulation. Parallel studies were conducted with human inner-wall SC cells isolated by a novel enzyme-assisted technique and with cannula-derived mixed inner- and outer-wall SC cells. A1 agonists increased whole cell currents of both inner-wall and cannula-derived SC cells. An A2A agonist reduced currents most consistently in specifically inner-wall SC cells. Those currents were also increased by A2B, but not consistently affected by A3, stimulation. A1, A2A, and A3 agonists all increased SC-cell intracellular Ca2+. The electrophysiological results are consistent with the possibility that inner-wall SC cells may mediate the previously reported modulatory effects of adenosine on outflow resistance. The results are also consistent with the presence of functional A2B, as well as A1, A2A, and A3 adenosine receptors in SC cells.

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. To address this question, excised patches and whole native bovine PE cells were patch clamped, and volume was monitored by calcein fluorescence. In symmetrical 130 mM NaCl, cAMP at the cytoplasmic surface of inside-out patches produced concentration-dependent activation of maxi-Cl(-) channels with a unitary conductance of 272 +/- 2 pS (n = 80). Voltage steps from 0 to +/-80 mV, but not to +/-40 mV, produced rapid channel inactivation consistent with the typical characteristics of maxi-Cl(-) channels. cAMP also activated the maxi-Cl(-) channels in outside-out patches. In both cases, maxi-Cl(-) channels were reversibly inhibited by SITS and 5-nitro-2-(phenylpropylamino)benzoate (NPPB). Decreasing cytoplasmic Cl(-) concentration reduced both open-channel probability and unitary conductance. Similarly, the membrane-permeant 8-bromo-cAMP stimulated outward and inward whole cell currents; the stimulation was larger at higher intracellular Cl(-) concentration. As with unitary currents, cAMP-triggered whole cell currents displayed inactivation at +/-80 but not at +/-40 mV. Moreover, cAMP triggered NPPB-sensitive shrinkage of PE cells. The results suggest that cAMP directly activates maxi-Cl(-) channels of native PE cells that contribute to Cl(-) release particularly from Cl(-)-loaded cells. These cAMP-activated channels provide a potential mechanism for reducing and modulating net aqueous humor secretion by facilitating Cl(-) reabsorption into the ciliary stroma.