Kim Peterson-Yantorno

Association of ClC-3 channel with Cl- transport by human nonpigmented ciliary epithelial cells.

Abstract. Electrophysiologic and volumetric evidence link the swelling-activated Cl− channels [gCl(Vol)] of nonpigmented ciliary epithelial (NPE) cells with the Cl−- channel/Cl−-channel regulator protein pICln. However, inhibitors (verapamil and dideoxyforskolin) of another Cl− channel/regulator (MDR1) have been found to inhibit the volume-activated transport response [the regulatory volume decrease (RVD)] of bovine NPE cells. We have addressed the possible molecular basis for the NPE Cl− channels by volumetric measurements of ODM human NPE cells in hypotonic and isotonic test solutions, and by polymerase chain reaction (PCR) cloning and Northern analyses of the same cells. Verapamil and dideoxyforskolin did inhibit the RVD. However, at a concentration (100 μm) which blocks >90% of the MDR1-associated Cl− currents, forskolin had no effect on the volume-activated Cl− channels or on the inhibition of those channels by protein kinase C. High concentrations of ATP (3.5 and 10 mm) and niflumic acid (IC50∼ 200 μm) also block [gCl(Vol)]. The RVD is inhibited by 9-phenylanthranilic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), unaffected by anthracene-9-carboxylic acid (9-AC), and stimulated by ionomycin. The Cl−-channel blockers NPPB, niflumic acid, DPC and 9-AC, and the Ca2+-ionophore ionomycin had qualitatively similar effects on the rate of staurosporine-activated isotonic cell shrinkage. These results support the concept that the volume-sensitive protein pICln regulates the Cl− channels, and that the same conduits subserve volume- and staurosporine-activated Cl− release. Of the cloned and sequenced Cl− channels, ClC-3 uniquely conforms to the stationary currents and PKC sensitivity of the NPE Cl− channels. PCR amplifications of human cDNA libraries from ciliary body, NPE cells and retina with primers based on human ClC-3 and ClC-4 cDNA, and Northern analyses using the products generated indicated that ciliary epithelial cells express transcripts for ClC-3 (but not ClC-4). We suggest that ClC-3 provides the same conduit for both volume-activated and isotonically staurosporine-activated Cl− channels of human nonpigmented ciliary epithelial cells.

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-μ M 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.

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+. Adenosines actions were further studied with nonpigmented ciliary epithelial (NPE) cells from continuous human HCE and ODM lines and freshly dissected bovine cells. With gramicidin present, adenosine (≥3 μM) triggered isosmotic shrinkage of the human NPE cells, which was inhibited by the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) and niflumic acid. At 10 μM, the nonmetabolizable analog 2-chloroadenosine and AMP also produced shrinkage, but not inosine, UTP, or ATP. 2-Chloroadenosine (≥1 μM) triggered increases of whole cell currents in HCE cells, which were partially reversible, Cl- dependent, and reversibly inhibited by NPPB. Adenosine (≥10 μM) also stimulated whole cell currents in bovine NPE cells. We conclude that occupancy of adenosine receptors stimulates Cl- secretion in mammalian NPE cells.

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

A1 adenosine receptors (ARs) reduce, and A2ARs increase intraocular pressure, partly by differentially altering resistance to aqueous humor outflow. It is unknown whether the opposing effects of A1AR and A2AR 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 A1, A2Aa and A3AR agonists. Receptor activation in human TM cells was identified by applying subtype-selective AR agonists: CPA and ADAC for A1 ARs, CGS 21680 and DPMA for A2AARs, and Cl-IB-MECA and IB-MECA for A3ARs. Stimulation of A1 A2A and A3ARs elevated Ca2+, measured with fura-2. Whole-cell patch clamping indicated that AR agonists activated ion channels non-uniformly, possibly reflecting variability in magnitude of agonist-triggered second-messenger responses. A], A2A and A3AR agonists all reduced volume, determined by calcein cell imaging. The endogenous source of adenosine delivery to the outflow pathway could be the TM cells since these cells were stimulated to release ATP by hypotonic perfusion. We conclude that: (1) TM cells express functional A1, A2A and A3ARs; and (2) the reported differential effects of AR agonists on aqueous humor outflow are not mediated by differential actions on TM-cell Ca2+ and volume, but likely by actions on separate cell targets.

Regulatory volume decrease by cultured non-pigmented ciliary epithelial cells.

Cells (ODM C1-2/SV40) derived from human non-pigmented ciliary epithelial cells were studied by electronic cell sizing. The time course of the cell volume (vc) was monitored after suspending cells in paired experimental and control, isosmotic and hyposmotic solutions of identical ionic composition. Following anisosmotic cell swelling, the cells displayed the regulatory volume decrease (RVD) previously described. The RVD primarily reflects loss of cell KCl since: (1) the K(+)-channel blockers quinidine and Ba2+ both inhibit the RVD; and (2) replacement of external Cl- with gluconate or addition of the Cl- channel blocker NPPB also inhibits the RVD. Bicarbonate has previously been reported to speed the RVD. This action likely reflects pH dependence of the channels since: (1) increasing the external pH speeds the RVD, whether or not HCO3- is present; and (2) DIDS (a blocker of Cl- channels and of Cl-/HCO3- exchange) is an effective inhibitor of the RVD, even after blocking Cl-/HCO3- exchange by removing external HCO3-. The RVD could also be inhibited by reducing the availability of Ca2+, either by omitting Ca2+ from the external medium or by blocking mobilization of intracellular Ca2+ with TMB-8. Furthermore, the RVD was slowed and incomplete in the presence of the calcium/calmodulin blocker trifluoperazine. We conclude that anisosmotic swelling triggers a series of events, mediated at least in part by calcium/calmodulin, leading to the extrusion of KCl through parallel K+ and Cl- channels.

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). Net formation of aqueous humor must, however, be the algebraic sum of unidirectional secretion and unidirectional reabsorption from the aqueous humor back into the stoma. The mechanisms potentially underlying reabsorption of aqueous humor by the NPE cells have recently been addressed by studying the regulatory response (RVI) of anisosmotically shrunken NPE cells. The results indicated that epithelial Na+ channels with a high affinity to amiloride likely contribute to reabsorption of solute from the aqueous humor. We have substantiated this possibility by using Northern analysis to identify in human ciliary body RNA a 3.7-kb transcript corresponding to the alpha-subunit of the amiloride-sensitive, alpha beta gamma-ENaC epithelial sodium channel. We have also found that the Na(+)-channel inhibitor benzamil inhibits the RVI without affecting the cell volume of isotonic cell suspensions. This observation supports the hypothesis that the low conductance, highly selective epithelial Na+ channel is activated by shrinkage and contributes to unidirectional reabsorption as aqueous humor. Examples are provided of how the integrative regulation of aqueous humor formation can involve conjugate actions on both unidirectional secretion and reabsorption.

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 A1 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 P2X7 receptors (P2RX7) 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 P2RX7. ATP release was also triggered by raising intracellular Ca2+ activity with ionomycin after a prolonged lag time and was unaffected by the PX1 blocker probenecid, but nearly abolished by P2RX7 antagonists. We conclude that swelling‐stimulated ATP release from human TM cells is physiologically mediated by PX1 and Cx hemichannels and P2X7 receptors, but not by vesicular release. PX1 appears not to be stimulated by intracellular Ca2+ in TM cells, but can be modulated by oxidation‐reduction state. The P2RX7‐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. J. Cell. Physiol. 227: 172–182, 2012.

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.

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 A3ARs in the ciliary epithelium activate Cl- channels, favoring increased formation of aqueous humor and elevated IOP. In contrast, stimulating A1ARs 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 P2X7 release pathways were functionally limited to inflow and outflow cells, respectively, suggesting that blocking exocytosis might selectively inhibit inflow, lowering IOP.

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.