Purines can modify ciliary epithelial secretion of aqueous humor into the eye. The source of the purinergic agonists acting in the ciliary epithelium, as in many epithelial tissues, is unknown. We found that the f luorescent ATP marker quinacrine stained rabbit and bovine ciliary epithelia but not the nerve fibers in the ciliary bodies. Cultured bovine pigmented and nonpigmented ciliary epithelial cells also stained intensely when incubated with quinacrine. Hypotonic stimulation of cultured epithelial cells increased the extracellular ATP concentration by 3-fold; this measurement underestimates actual release as the cells also displayed ecto-ATPase activity. The hypotonically triggered increase in ATP was inhibited by the Cl ؊ -channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in both cell types. In contrast, the P-glycoprotein inhibitors tamoxifen and verapamil and the cystic fibrosis transmembrane conductance regulator (CFTR) blockers glybenclamide and diphenylamine-2-carboxylate did not affect ATP release from either cell type. This pharmacological profile suggests that ATP release is not restricted to P-glycoprotein or the cystic fibrosis transmembrane conductance regulator, but can proceed through a route sensitive to NPPB. ATP release also was triggered by ionomycin through a different NPPB-insensitive mechanism, inhibitable by the calcium͞calmodulin-activated kinase II inhibitor KN-62. Thus, both layers of the ciliary epithelium store and release ATP, and purines likely modulate aqueous humor f low by paracrine and͞or autocrine mechanisms within the two cell layers of this epithelium.Glaucoma, a major cause of blindness, usually is associated with elevated intraocular pressure. Pharmacologic treatment of the ocular hypertension is now largely directed toward reducing aqueous humor formation by the ciliary epithelium, a bilayer consisting of a nonpigmented (NPE) and a pigmented (PE) epithelial cell layer (1). The precise mechanisms underlying aqueous humor secretion into the eye are uncertain, but the release of Cl Ϫ through basolateral channels of the NPE cells is thought to be rate limiting (2, 3). Thus, agents that can alter this Cl Ϫ conductance either directly or indirectly will alter aqueous humor production.Several recent observations suggest important roles for extracellular ATP and adenosine in the regulation of ciliary epithelial chloride conductance and in the formation of aqueous humor. Purinergic receptors for adenosine and ATP are expressed by both the NPE and PE cells of the ciliary epithelial bilayer (4). Adenosine modifies the transport of Cl Ϫ in the NPE cells (5) and alters the flow of aqueous humor (6, 7), and ATP affects Cl Ϫ transport in the PE cells (8). Purines are detectable in the aqueous humor, and the combined concentrations of adenosine and its metabolite inosine can rise to micromolar levels (9). Although purines appear to be major regulators of aqueous humor formation, their physiological source is unknown. This study sought to identify the source and...

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. The A3-selective agonist N 6-(3-iodobenzyl)-adenosine-5′- N-methyluronamide (IB-MECA) triggered shrinkage (apparent K d = 55 ± 10 nM). A3-selective antagonists blocked IB-MECA-triggered shrinkage, and A3-antagonists (MRS-1097, MRS-1191, and MRS-1523) also abolished shrinkage produced by 10 μM adenosine when all four known receptor subtypes are occupied. The A1-selective agonist N 6-cyclopentyladenosine exerted a small effect at 100 nM but not at higher or lower concentrations. The A2A agonist CGS-21680 triggered shrinkage only at high concentration (3 μM), an effect blocked by MRS-1191. IB-MECA increased intracellular Ca2+ in HCE cells and also stimulated short-circuit current across rabbit ciliary epithelium. A3 message was detected in both HCE cells and rabbit ciliary processes using RT-PCR. We conclude that human HCE cells and rabbit ciliary processes possess A3 receptors and that adenosine can activate Cl− channels in NPE cells by stimulating these A3receptors.

1. Glaucoma is a worldwide disease affecting approximately 1-2% of the population aged over 35 years in industrial countries and is a major cause of blindness. 2. Glaucoma is usually associated with an increased intraocular pressure reflecting an imbalance between the rate of production of fluid (the aqueous humor) by the ciliary epithelial cells and its drainage from the eye. Therefore, it is important to understand how this secretion is produced. This requires a knowledge of ciliary epithelial cell composition, which has, in the past, proved difficult to obtain in mammalian preparations. 3. We have recently used the technique of electron-probe X-ray microanalysis to determine this composition under a variety of in vitro conditions. 4. Our results have led to a new model for this secretion that emphasizes the potential secretory role of the Na+/K+/2Cl- cotransporter.

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. 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.