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...
Form-deprivation myopia, in its early stages, is associated with only minimal changes in retinal gene expression at the level of the transcriptome. While the list of validated genes is short, each merits further study for potential involvement in the signaling cascade mediating myopia development.
Plus or minus lens wear induce markedly different, not opposite, alterations in retina/RPE gene expression. The initial retinal responses to defocus are quite different from those when the eye growth patterns are well established, suggesting that different mechanisms govern the initiation and persistence or progression of refractive errors. The gene lists identify promising signaling candidates and regulatory pathways for future study, including a potential role for circadian rhythms in refractive development.
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.
Stimulation of ATP or adenosine receptors causes important physiological changes in retinal pigment epithelial (RPE) cells that may influence their relationship to the adjacent photoreceptors. While RPE cells have been shown to release ATP, the regulation of extracellular ATP levels and the production of dephosphorylated purines is not clear. This study examined the degradation of ATP by RPE cells and the physiological effects of the adenosine diphosphate (ADP) that result. ATP was readily broken down by both cultured human ARPE-19 cells and the apical membrane of fresh bovine RPE cells. The compounds ARL67156 and ␥-mATP inhibited this degradation in both cell types. RT-PCR analysis of ARPE-19 cells found mRNA message for multiple extracellular degradative enzymes; ectonucleotide pyrophosphatase/phosphodiesterase eNPP1, eNPP2, and eNPP3; the ectoATPase ectonucleoside triphosphate diphosphohydrolase NTPDase2, NTPDase3, and some message for NTPDase1. Considerable levels of ADP bathed RPE cells, consistent with a role for NTPDase2. ADP and ATP increased levels of intracellular Ca 2ϩ . Both responses were inhibited by thapsigargin and P2Y1 receptor inhibitor MRS 2179. Message for both P2Y1 and P2Y12 receptors was detected in ARPE-19 cells. These results suggest that extracellular degradation of ATP in subretinal space can result in the production of ADP. This ADP can stimulate P2Y receptors and augment Ca 2ϩ signaling in the RPE.ectoapyrase; PC-1; CD39; CD39L1; P2Y1; P2Y 12 ; ADP; ATP release; photoreceptors; retinal detachment THE RETINAL PIGMENT EPITHELIUM (RPE) has an intimate anatomic relationship with the photoreceptor outer segments. This juxtaposition underlies a close functional relationship, and the RPE performs a variety of roles that maximize photoreceptor health. For example, the distal tips of the outer segments are regularly phagocytosed by the RPE cells to enable continual resynthesis of these outer segments (37). Transport mechanisms on the apical membrane of the RPE regulate the ionic composition of the subretinal space located between the RPE cells and the outer segments, controlling the ionic driving forces on the photoreceptor outer segments (15). In addition, the transport of fluid and ions from the apical membrane facing the photoreceptors to the basolateral membrane of the RPE is thought to be one of the main forces keeping the retina attached (21).The exogenous addition of purines can trigger responses capable of modifying the interaction between the RPE and photoreceptors. Stimulation of ATP receptors increases the rate of ion and fluid transport from subretinal space toward the choroid (30). Agonists for P2Y 2 receptors can increase Ca 2ϩ levels in RPE cells and consequently enhance the rate of fluid absorption across monolayers of bovine and fetal human RPE cells. This stimulation may have important clinical implications because agonists facilitate retinal reattachment in rat (20) and rabbit models of retinal detachment (25). The dephosphorylated nucleoside adenosine can also modify the r...
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