Nonpigmented Ciliary Epithelial Cells Research Articles

A release mechanism for stored ATP in ocular ciliary epithelial cells.

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 fluorescent 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 calcium/calmodulin-activated kinase II inhibitor KN-62. Thus, both layers of the ciliary epithelium store and release ATP, and purines likely modulate aqueous humor flow by paracrine and/or autocrine mechanisms within the two cell layers of this epithelium.

Endothelin receptor A is expressed and mediates the [Ca2+]i mobilization of cells in human ciliary smooth muscle, ciliary nonpigmented epithelium, and trabecular meshwork

PURPOSE. To identify which endothelin receptor subtype is expressed and is functional in the human ciliary body and trabecular meshwork, tissues that regulate aqueous humor dynamics. METHODS. Immunocytochemistry was used to characterize the primary culture cells of normal human ocular cells. Endothelin receptor gene expression was probed with reverse transcription of polymerase chain reaction (RT-PCR). Intracellular calcium ([Ca 2+ ] i) mobilization was measured with video image microscopy using Fura-2AM as a fluorescent probe. RESULTS. Identities of primary cultures, human ciliary smooth muscle (HCSM), ciliary nonpigmented epithelial (HCE), and trabecular meshwork (HTM) cells were confirmed by immuno-cytochemistry, using cell-specific markers and observing typical cell morphologies. The presence of endothelin receptor A (ET A) was detected with RT-PCR in all three types of cells. The mRNA phenotype was verified with restriction enzyme BamHI digestion. No ET B receptor subtype expression was detected with RT-PCR under the cell culture conditions used. The [Ca 2+ ] i of HCSM cells was increased from 57 ± 7 nM to 328 ± 108 nM (n = 23; mean ± SE; P < 0.05) by 1 nM endothelin-1 (ET-1). In HCE cells, [Ca 2+ ] i increased from 40 ± 3 nM to 90 ± 10 nM (n = 55) (P < 0.001) with the same concentration of ET-1. Similarly, ET-1 (1 nM) increased the [Ca 2+ ] i from 51 ± 6 nM to 185 ± 47 nM (n = 19) (P < 0.001) in the HTM cells. The agonist for ET B, S6c, had no effect on [Ca 2+ ] i transients in all three cell types. No ET B receptor expression was detected in these cell types under the experimental and culture conditions. CONCLUSION. ET A receptor is expressed and is possibly responsible for mediating the signal for [Ca 2+ ] i mobilization by ET-1 in human ciliary smooth muscle, ciliary nonpigmented epithelial cells, and trabecular meshwork cells.

Iridociliary epithelial tumors in 100 dogs and 17 cats: a morphological study.

The morphological features of iridociliary epithelial tumors in 100 dogs and 17 cats were reviewed. Twenty-seven cases were in either Golden Retrievers or Labrador Retrievers. Affected globes were stained for light microscopy with alcian blue, periodic acid Schiff (PAS) and hematoxylin and eosin stains. Selected tissues were examined by immunohistochemistry for vimentin, desmin, cytokeratin, S-100, neuron-specific enolase (NSE), and glial fibrillary acid protein (GFAP). The presence or absence of hyaluronic acid was recorded by staining with alcian blue before and after digestion of the tissue with hyaluronidase. Canine tumors were divided into papillary and solid tumors based on the pattern of growth. Twenty-eight of 57 papillary tumors exhibited invasive behavior including eight of the 57 which showed infiltration of the sclera. Twenty-nine of 43 solid tumors were invasive including 13 of 43 with scleral invasion. Tumors with scleral invasion were designated adenocarcinoma. Tumors of both types could be pigmented or nonpigmented and often contained smooth basement membranes reminiscent of the inner membrane of the nonpigmented ciliary body epithelial cell. All of the feline tumors were nonpigmented and 14 of 16 feline tumors were solid and two of the tumors were papillary. Eighteen of 20 canine tumors and three of four feline tumors stained positive for vimentin. Cytokeratin stain was positive only in a few of the highly aggressive tumors. The finding of pigmented epithelial cells, thick, smooth basement membrane structures, positive staining for vimentin, S-100, and NSE as well as hyaluronic acid deposition were considered to be features which define iridociliary epithelial tumors in dogs. The positive staining for vimentin and NSE are highly specific markers which help to characterize iridociliary epithelium and distinguish this tumor from metastatic epithelial tumors. The finding of solid nonpigmented tumors with small epithelial cells packeted by thin PAS-positive membranes staining positive for vimentin were considered significant features defining iridociliary epithelial tumors in cats. Follow-up information on survival and cause of death was obtained on 43 canine cases and only two feline cases. The average follow-up interval in dogs was 25 months and only two dogs died with lesions that could have been due to metastasis although neither was confirmed. We concluded that neither iridociliary adenomas nor adenocarcinomas is likely to metastasize.

Regulation of Endothelin-1 in Human Non-Pigmented Ciliary Epithelial Cells by Tumor Necrosis Factor-α

Endothelins (ET) are potent vasoactive peptides present in many ocular structures and are formed from precursor Big endothelins (Big ET-1) by the action of an endothelin-converting enzyme (ECE). ET-1 is thought to decrease intraocular pressure by contracting the ciliary muscle thus enhancing the outflow of aqueous humor through the Canal of Schlemm and trabecular meshwork. However, the mechanisms involved in the regulation of endothelin-1 (ET-1) synthesis and release in ocular tissues have not been fully characterized. In this study we examined the effect of tumor necrosis factor-α(TNF-α; 10 nm), a proinflammatory cytokine, on the cellular mechanisms leading to ET-1 synthesis and release in SV-40 transformed human ciliary non-pigmented epithelial cells (HNPE). ET-1 and Big endothelin-1 (Big ET-1) immunoreactivity was time-dependently increased following TNF-αtreatment. Phorbol esters (PMA), activators of PKC, also raised the immunoreactive levels of ET-1 and Big ET-1 while, staurosporine, a PKC inhibitor (20 nm), decreased ET-1 levels in TNF-α-stimulated cells. Pre-treatment with phosphoramidon (1 μm) an ECE-inhibitor, followed by TNF-αstimulation, decreased ir-ET-1 levels. Cycloheximide (9 μm), a protein synthesis inhibitor, decreased TNF-α-stimulated levels for ir-ET-1 and ir-Big ET-1, suggesting that TNF-αmay be directly regulating ET-1 expression at the ET-1 gene. Our data indicates that TNF-α regulates ET-1 levels in HNPE cells possibly by activating PKC either to stimulate protein synthesis and/or to enhance ET-1 secretion. These results suggest that ET-1 released from the ciliary body may play an important role in aqueous humor dynamics following cytokine activation.

Functional coupling in bovine ciliary epithelial cells is modulated by carbachol.

The functional coupling of the ciliary epithelium was studied in isolated pairs (couplets) of pigmented ciliary epithelial (PCE) and nonpigmented ciliary epithelial (NPCE) cells using the whole cell patch clamp and the fluorescent dye lucifer yellow. One cell of the pair (usually the NPCE cell of a NPCE-PCE cell couplet) was accessed with a 2-5 M omega electrode, containing 1-2 mM lucifer yellow, in the whole cell configuration of the patch clamp. After voltage-clamp experiments were completed, cells were viewed under a fluorescent microscope to confirm that the cells were coupled. The electrical coupling of the cells was also studied by calculating the capacitance (using the time-domain technique), assuming a "supercell" model for coupled cells. The mean capacitance of coupled pairs was 79.8 +/- 4.3 (SE) pF (n = 47) compared with single cell capacitances of 36.8 +/- 3.4 pF (n = 10) for PCE cells and 38.1 +/- 3.1 pF (n = 15) for NPCE cells. Octanol, carbachol (CCh), and raised extracellular Ca2+ concentration ([Ca2+]o) all caused uncoupling in pairs (couplets) of coupled NPCE and PCE cells. At room temperature (22-24 degrees C), the capacitance of the couplets decreased from 70.5 +/- 8.0 to 48.0 +/- 5.2 pF (n = 5) when exposed to octanol (1 mM), from 73.8 +/- 9.2 to 43.2 +/- 9.5 pF (n = 4) when exposed to CCh (100 microM), and from 80.5 +/- 6.7 to 49.9 +/- 7.8 pF (n = 4) when exposed to 10 mM [Ca2+]o. The response to CCh was dose dependent; at higher temperatures of 34-37 degrees C, 10 microM CCh caused a 38% reduction in capacitance, from 53.7 +/- 9.7 to 33.5 +/- 3.3 pF (n = 7) with a half-time of 249 s, and 100 microM CCh caused a 49% reduction in capacitance, from 51.3 +/- 5.6 to 26.0 +/- 2.4 pF (n = 7) with a half-time of 124 s. After pairs uncoupled and the uncoupling agent was washed out, the cell pairs often exhibited an increase in capacitance that we interpreted as "recoupling" or a reopening of the gap junctional communication pathway; the half-time for this process was 729 s after uncoupling with 100 microM CCh and 211 s after uncoupling with 10 microM CCh. This interpretation was confirmed optically by the spread of lucifer yellow into both cells of an uncoupled pair with a time course corresponding to the increase in electrical coupling. The controllable coupling of ciliary epithelial cells extends the idea of a functional syncytium involved in active transport. PCE cells take up solute and water from the blood, which then cross to NPCE cells via gap junctions and from there are secreted into the posterior chamber of the eye. Modulation of the coupling between NPCE and PCE cells may provide a mechanism to control secretion.

Influence of bafilomycin A1 on pHi responses in cultured rabbit nonpigmented ciliary epithelium.

Aqueous humor secretion is in part linked to HCO3- transport by nonpigmented ciliary epithelium (NPE) cells. During this process, the cells must maintain stable cytoplasmic pH (pHi). Because a recent report suggests that NPE cells have a plasma membrane-localized vacuolar H(+)-ATPase, the present study was conducted to examine whether vacuolar H(+)-ATPase contributes to pHi regulation in a rabbit NPE cell line. Western blot confirmed vacuolar H(+)-ATPase expression as judged by H(+)-ATPase 31-kDa immunoreactive polypeptide in both cultured NPE and native ciliary epithelium. pHi was measured using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Exposing cultured NPE to K(+)-rich solution caused a pHi increase we interpret as depolarization-induced alkalinization. Alkalinization was also caused by ouabain or BaCl2. Bafilomycin A1 (0.1 microM; an inhibitor of vacuolar H(+)-ATPase) inhibited the pHi increase caused by high K+. The pHi increase was also inhibited by angiotensin II and the metabolic uncoupler carbonyl cyanide m-chlorophenylhydazone but not by ZnCl2, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), omeprazole, low-Cl- medium, HCO3(-)-free medium, or Na(+)-free medium. Bafilomycin A1 slowed the pHi increase after an NH4Cl (10 mM) prepulse. However, no detectable pHi change was observed in cells exposed to bafilomycin A1 under control conditions. These studies suggest that vacuolar H(+)-ATPase is activated by cytoplasmic acidification and by reduction of the proton electrochemical gradient across the plasma membrane. We speculate that the mechanism might contribute to maintenance of acid-base balance in NPE.

Molecular cloning and expression of a chloride channel-associated protein pICln in human young red blood cells: association with actin.

We report the cloning and sequencing from human reticulocytes of cDNA coding for the Cl- channel-associated protein, pICln. Human reticulocyte pICln (HRpICln) cDNA encodes a protein (predicted molecular mass 26293Da) identical with human non-pigmented ciliary epithelial cell pICln. By using full-length HRpICln cDNA (approx. 1.2 kb) to probe human lymphocyte metaphase-chromosome spreads, the location of the human ICln gene was mapped to 11q13 by fluorescence in situ hybridization analysis. Polyclonal antibodies to recombinant HRpICln detected bands at approx. 43 kDa and approx. 37 kDa in both normal (AA) and sickle (SS) red blood cell (RBC) ghost membranes. In SS ghosts, and in ghosts from a patient with autoimmune haemolytic anaemia with 9.8% reticulocytes, the amount of HRpICln was increased compared with AA ghosts, suggesting that the expression or membrane assembly of HRpICln is cell age-dependent. Laser scanning confocal fluorescent microscopy immunolocalized HRpICln largely to the RBC membrane. The increased staining intensity of HRpICln in a reticulocyte-enriched AA RBC density-separated fraction is consistent with a dependence of HRpICln membrane content on cell age. HRpICln and beta-actin form stable complexes in vivo, demonstrated with the yeast two-hybrid system. Low-ionic-strength extraction of ghost membranes, which results in the extraction of the spectrin-actin cytoskeleton, also results in the extraction of HRpICln, consistent with the possibility for the association of these proteins in RBCs in vivo. The results presented here establish the presence of the Cl- channel-associated protein, pICln, in human RBCs, and raises the possibility that this protein has a role in RBC Cl- transport and volume regulation in young RBCs. Moreover the association of RBC pICln with actin offers a model in which to test interactions between RBC ion channels and the cytoskeleton.

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. With gramicidin present, adenosine (> or = 3 microM) 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 microM, the nonmetabolizable analog 2-chloroadenosine and AMP also produced shrinkage, but not inosine, UTP, or ATP. 2-Chloroadenosine (> or = 1 microM) triggered increases of whole cell currents in HCE cells, which were partially reversible, Cl- dependent, and reversibly inhibited by NPPB. Adenosine (> or = 10 microM) also stimulated whole cell currents in bovine NPE cells. We conclude that occupancy of adenosine receptors stimulates Cl- secretion in mammalian NPE cells.

Cloning and Functional Expression of a Swelling-Induced Chloride Conductance Regulatory Protein, plCln, from Rabbit Ocular Ciliary Epithelium

The cDNA encoding a swelling-induced chloride conductance regulatory protein, plcln, was cloned from rabbit ciliary epithelium by using a polymerase chain reaction (PCR)-based approach. The open reading frame encoding 236 amino acids possesses high amino acid identity (93/%) with the previously cloned plclnfrom human ciliary epithelium. Outwardly rectifying currents were recorded in Xenopus oocytes injected with plclncRNA, a result consistent with plclnexpression in ciliary epithelium. A widespread distribution and marked expression of plclnmRNA in both nonpigmented ciliary epithelial (NPE) cells and pigmented ciliary epithelial (PE) cells was found for the first time. In situ hybridization analysis showed that plclnexpression is more abundant in NPE than PE. These findings are consistent with the idea that plclnmay be an important regulatory element in these secretory cells.

Gene Expression of Proteases and Protease Inhibitors in the Human Ciliary Epithelium and ODM-2 Cells

Complementary DNAs (cDNAs), corresponding to the human proteinases cathepsins D and O and proteinase inhibitors α2-macroglobulin and PP5/TFPI-2, have recently been isolated and identified from a subtractive human ciliary body library. In the present study we determined: (i) their pattern of expression in the human eye; (ii) the ability of the ciliary body and/or ciliary epithelial cells to synthesize and secrete cathepsin D and α1-antitrypsin in vitro; and (iii) whether α1-antitrypsin expression in cultured ciliary epithelial cells is modulated by protein kinase C activation. Northern analysis demonstrated that the ciliary body expresses high levels of cathepsins D and O, α2-macroglobulin, α1-antitrypsin and PP5/TFPI-2 transcripts. Western blot analysis and immunoprecipitation experiments with cathepsin D and α1-antitrypsin antibodies indicated that metabolically labeled ciliary body explants and/or ciliary epithelial cells in vitro with35S-methionine, synthesize and secrete these proteins. Cultured nonpigmented ciliary epithelial ODM-2 cells, in response to phorbol-12-myristate 13-acetate (PMA), but not to the non-protein kinase C binding phorbol ester 4 α-phorbol didecanoate (PDBu), elicited up-regulation (up to 5-fold) of transcription, synthesis and secretion of α1-antitrypsin. These results provide in vitro evidence that the ciliary epithelium synthesizes and secretes a selective group of proteinases and proteinase inhibitors detected also in aqueous humor. The expression of at least of one of the proteinase inhibitors, α1-antitrypsin, can be modulated in response to phorbol ester.

Functional and morphological differentiation of nonpigmented ciliary body epithelial cells grown on collagen rafts

We have examined the effect of alteration in cell shape on promoting differentiated morphology and physiology in cultured nonpigmented epithelial cells from the ciliary body. We have grown pure populations of nonpigmented cells on collagen gels released from the culture dish to create collagen rafts. Shortly after the gels were detached, the cells shrank in diameter and increased in height while they contracted the gel. Concurrently, the actin cytoskeleton reorganized to the cell cortex as found in vivo. After this differentiated morphology developed, large changes in intracellular Ca2+ could be elicited by simultaneous activation of acetylcholine and epinephrine or acetylcholine and somatostatin receptors as seen in intact tissue. Explant cultures of isolated nonpigmented cell layers maintained their actin distribution and also showed synergistic Ca2+ increases. Spread cells, grown on rigid substrates, had a disorganized cytoskeleton and rarely showed synergism. These data suggest that the mechanism underlying synergistic Ca2+ responses in the ciliary body is functional in nonpigmented cells grown on collagen rafts. In addition, this pathway appears to be sensitive to the disposition of the cell's cytoarchitecture.

Differential regulation of natriuretic peptide receptors on ciliary body epithelial cells.

Atrionatriuretic peptide (ANP) lowers intraocular pressure in the eyes of humans and rabbits. We examined the effects of natriuretic peptides on cGMP formation and 125I-labelled-ANP binding to cultured cells derived from ciliary body epithelium, the site of aqueous humour formation in the eye. ANP, brain natriuretic peptide (BNP) and C-natriuretic peptide (CNP) at 1 microM stimulated cGMP formation 8.2(+/-1.2)-fold, 4.8(+/-0.6)-fold and 87.3(+/-12.1)-fold respectively. 125I-ANP bound to intact cells at a single site, with a dissociation constant KD=0.30+/-0.01 nM. BNP was as effective as ANP in displacing 125I-ANP, whereas CNP displaced label with a slightly higher IC50. 125I-ANP binding was displaced >95% by c-ANP, a specific ligand for natriuretic peptide C receptors (NPR-C). Cross-linking of 125I-ANP to cells labelled predominantly a protein of Mr 62000. These data suggest that 125I-ANP binding was primarily to NPR-C, whereas cGMP stimulation occurred primarily via natriuretic peptide B receptors (NPR-B). Vasopressin and histamine, both activators of the inositol phosphate/diacylglycerol phosphate pathway in non-pigmented ciliary epithelial cells, inhibited CNP stimulation of guanylate cyclase (NPR-B) and 125I-ANP binding (NPR-C) by 30-38%. Inhibition was mimicked by PMA, dioctanoylglycerol and phorbol didecanoate, whereas 4alpha phorbol didecanoate had no effect. Staurosporine and bisindolylmaleimide both blocked inhibition of 125I-ANP binding and cGMP formation by PMA. These results suggest that protein kinase C (PKC) down-regulates both NPR-B and NPR-C. PKC down-regulation of NPR-B varied inversely with CNP concentration. Inhibition by 1 microM PMA was 30.6(+/-4.0)% with 500 nM CNP, but 83.4(+/-8.8)% with 10 nM CNP, indicating that increasing CNP could partially overcome inhibition by PMA. Since extracellular CNP levels were not affected by PKC activation, the effect of PKC on NPR-B is best explained as a reduction in NPR-B affinity for CNP. NPR-C measured as 125I-ANP binding was likewise reduced 36.4(+/-5.1)% by exposure to PMA. In contrast with NPR-B inhibition, however, inhibition of NPR-C was due largely to a reduction in the number of receptor binding sites per cell rather than a reduction in receptor affinity for ligand. The data therefore suggest that both NPR-B and NPR-C are down-regulated by PKC, but that the mechanisms of down-regulation of the two receptors are different.

Detection of EP 2, EP 4, and FP receptors in human ciliary epithelial and ciliary muscle cells

We have examined the expression of three prostaglandin (PG) receptors, EP 2, EP 4, and FP, in a nonpigmented ciliary epithelial cell line (ODMC1-2) and in human ciliary muscle (HCM) cells. Total RNA preparations from either ODMCl-2 or HCM cells were subjected to reverse transcription-polymerase chain reaction (RT-PCR) with sense and antisense primers for each of the three PG receptors. The RT-PCR generated DNA products of predicted sizes corresponding to the EP 2, EP 4, and FP receptors in both ODMCl-2 and HCM cells. PCR products corresponding to each receptor were hybridized with specific 32P-labeled probes and, for further confirmation, digested with appropriate restriction enzymes. Pharmacological studies with the EP 2 receptor-selective agonist butaprost resulted in a significant increase in the cyclic AMP level in ODMCl-2 cells. The stimulation of cyclic AMP in ODMCl-2 cells by PGE 2 and 11-deoxy PGE 1, the respective EP 1/EP 2/EP 3 EP4 and EP 2/EP 3 EP 4 receptor agonists, was concentration-dependently inhibited by the EP 4 receptor-selective antagonist AH23848. These results conclusively demonstrate the presence of both mRNA and protein for EP 2, EP 4, and FP receptors in ODMC1-2 and HCM cells.

Three different Cl- channels in the bovine ciliary epithelium activated by hypotonic stress.

1. Hypotonic solution induced transient Cl- channel activity in both pigmented and non-pigmented ciliary epithelial cells in cell-attached patches. 2. The activation time constants for these currents were 63 and 97 s for non-pigmented and pigmented ciliary epithelial (NPCE and PCE) cells, respectively. The currents inactivated during the exposure to hypotonic solution with time constants of 59 and 304 s for NPCE and PCE cells, respectively. 3. Single channel analysis revealed the presence of a low-conductance Cl- channel in both the NPCE (7.3 +/- 0.4 pS) and PCE (8.6 +/- 0.9 pS) cells. In addition, an intermediate-conductance (18.8 pS) channel was found alone in the NPCE cells and a maxi Cl- channel (105 pS) was found in the PCE cells. The similarities between the 18.8 pS Cl- channel and the volume-activated organic osmolyte-anion channel (VSOAC) are discussed. 4. The hypotonic activation of all three Cl- channels was prevented by the inclusion of 100 microM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in the patch pipette. 5. Inclusion of tamoxifen in the patch pipette inhibited only the intermediate-conductance channel in the NPCE cells. This is consistent with the identification of the intermediate 18 pS Cl- channel, found only in NPCE cells, as VSOAC. 6. Negative pressure also evoked single Cl- channel activity but the activation kinetics were quite different, suggesting that, even if the channels are the same, the second messenger pathways involved are different. 7. The activation/inactivation kinetics of these Cl- channels correlate well with the time course of regulatory volume decrease (RVD) in these cells, suggesting that these channels may well participate in the process of RVD. 8. The asymmetrical distribution of Cl- channels in the ciliary epithelium may be significant for aqueous humour secretion. For instance, the presence of the VSOAC-like channel in the NPCE cells may allow one-way traffic of anions and organic osmolytes into the eye and, if these solutes were loaded by the PCE cells, then a vectorial flow from the blood into the eye would occur.

The prostaglandin transporter is widely expressed in ocular tissues

Prostaglandins (PGs) play important physiological and therapeutic roles in the eye. Our laboratory recently identified a novel PG transporter in the rat that we call “PGT” (Science 268:866, 1995). We have also recently cloned the human PGT cDNA (J Clin Invest 98:1142, 1996). To determine whether PGT might play a role in human ocular tissues, we performed Northern blot analysis of RNA obtained from human ocular tissues and from the nonpigmented ciliary epithelium cell line “ODM-2.” PGT transcripts were clearly evident in all ocular tissues. Given that the functional profile of PGT expressed in vitro strongly suggests a role in PG uptake and degradation, the present results suggest that PGT may function in various regions of the human eye for purposes of terminating the signal(s) produced by locally-synthesized PGs.

Relationship between caffeine-induced ocular hypertension and ultrastructure changes of non-pigmented ciliary epithelial cells in rats.

The purpose of this study was to morphologically assess a possible mechanism for caffeine-induced ocular hypertension. Taking into consideration the relationship between the secretion of aqueous humor and the ultrastructure of the ciliary body, the time course of the morphological features in the ciliary epithelium when caffeine was administered intravenously to male Wistar rats was investigated by electron-microscopy. These morphological findings were also compared with the changes in the intraocular pressure (IOP). A significant increase in IOP was noted 15 min and 1 hr after a single dosing of caffeine alone. This change disappeared in all animals within 2 hr after dosing. The IOP in the animals receiving caffeine and the beta-blocker befunolol, which lowers the IOP by inhibiting aqueous humor secretion, decreased significantly from 15 min after dosing, and this change persisted 2 hr after dosing. In electron-microscopy 15 min and/or 1 hr after dosing with caffeine, a slight dilatation in the lateral intercellular spaces near the basement membrane of the non-pigmented ciliary epithelium was observed and the interdigitations between the non-pigmented epithelial cells were intact. Reversal of these changes was observed 2 hr after dosing. On the other hand, the lateral intercellular spaces between the non-pigmented epithelial cells were markedly dilated and the interdigitations were disorganized following dosing with caffeine alone and in combination with befunolol. These results described here indicate that the intravenous administration of caffeine causes ocular hypertension and also changes in the non-pigmented ciliary epithelium, suggesting an enhancement of aqueous humor transportation. This paradigm in the rat is considered to be useful to further assess caffeine-induced ocular hypertension and for use as an animal model in glaucoma research associated with an aqueous humor secretion.

Volume-sensitive chloride current in pigmented ciliary epithelial cells: role of phospholipases.

The whole cell recording technique was used to examine an outwardly rectifying chloride current activated by hypotonic shock in bovine pigmented ciliary epithelial (PCE) cells. Removal of internal and external Ca2+ did not affect the activation of these currents, but they were abolished by the phospholipase C inhibitor neomycin. The current was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and 4,4'-disothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner, but tamoxifen, dideoxyforskolin, and quinidine did not affect it. This blocking profile differs from that of the volume-sensitive chloride channel in neighboring nonpigmented ciliary epithelial cells (Wu, J., J. J. Zhang, H. Koppel, and T. J. C. Jacob, J. Physiol, Lond. 491: 743-755, 1996), and this difference implies that the volume responses of the two cell types are mediated by different chloride channels (Jacob, T. J. C., and J. J. Zhang. J. Physiol. Lond. In press). Intracellular administration of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to PCE cells induced a transient, time-independent, outwardly rectifying chloride current that closely resembled the current activated by hypotonic shock. DIDS produced a voltage-dependent block of the GTP gamma S-activated current similar to the block of the hypotonically activated current. Intracellular neomycin completely prevented activation of this current as did incubation of the cells in calphostin C. and inhibitor of protein kinase C (PKC). Removal of Ca2+ did not affect activation of the current by GTP gamma S but extended the duration of the response. Inhibition of phospholipase A2 (PLA2) with p-bromophenacyl bromide prevented the activation of the hypotonically induced current and also inhibited the current once activated by hypotonic solution. The findings imply that the hypotonic response in PCE cells is mediated by both phospholipase C (PLC) and PLA2. Both phospholipases generate arachidonic acid, and, in addition, the PLC pathway regulates the PLA2 pathway via a PKC-dependent phosphorylation of PLA2.

Comparative studies on prostanoid receptors in human non-pigmented ciliary epithelial and mouse fibroblast cell lines

To examine the expression of functional prostanoid receptors in the human non-pigmented ciliary epithelial (ODMC1–2) and mouse fibroblast cell lines (NIH 3T3) we have measured the generation of the second messengers, cyclic AMP, inositol phosphates and the mobilization of intracellular calcium ([Ca 2+] i) following stimulation by prostaglandin receptor agonists. The amount of cyclic AMP generated was measured by a protein binding method. Radiolabeled inositol phosphates were separated using ion exchange columns and quantified by counting the radioactivity. For intracellular calcium measurements, Fura 2-AM loaded cells were stimulated by PG receptor agonists and the calcium activated fluorescence was measured in a spectrofluorometer. In the ODMC1–2 cell line, the formation of cyclic AMP was stimulated by prostaglandin E 2, butaprost and 11-deoxy-prostaglandin E 1. The stimulation of cyclic AMP production by prostaglandin E 2 was partially inhibited by the EP 4 receptor antagonist AH23848. Prostaglandin E 2 and 11-deoxy-prostaglandin E 1 stimulated the formation of cyclic AMP in NIH 3T3 cells. In ODMC1–2 cells, total inositol phosphate turnover was not increased by 17-phenyl-trinor-prostaglandin F 2α, 17-phenyl-trinor-prostaglandin E 2 or 11-deoxy-prostaglandin E 1. In contrast, all these agonists, with the exception of 11-deoxy-prostaglandin E 1, significantly increased total inositol phosphates in NIH 3T3 cells. In the NIH 3T3 cell line, only prostaglandin F 2α and 17-phenyl-trinor-prostaglandin F 2α increased [Ca 2+] i in a dose-dependent manner; in ODMC1–2 cells, neither these agonists nor 17-phenyl-trinor-prostaglandin E 2 increased [Ca 2+] i. The present studies suggest that in ODMC1–2 cells, EP 2 and EP 4 receptors but neither EP 1 nor FP receptors are expressed; these studies also imply, NIH 3T3 cells express EP 2 and FP receptors, while EP 1 receptors appear to be absent in this cell line.

Elevation of intracellular Ca 2+ concentration in rabbit nonpigmented ciliary epithelial cells by allicin

A previous study has shown that allicin produces changes in aqueous humor dynamics, and this study was conducted to examine possible cellular mechanisms. In rabbit nonpigmented ciliary epithelial cells, basal levels of [Ca 2+], were determined to be 164 ± 34 nM. Allicin, a sulfhydryl-reactive agent, induced Ca 2+ transients at 0.01 mM and at 0.2 mM, the Ca 2+ transient peaked at 732 ± 35 nM. Allicin-induced Ca 2+ transients were prevented by pretreatment with dithiothreitol which did not affect the basal Ca 2+ levels. Allicin had only a slight, insignificant, effect on L-type Ca 2+ currents, and allicin-induced Ca 2+ transients were also present under extracellular Ca 2+-free conditions. These data suggest that intracellular Ca 2+ stores are the most probable source of allicin's effect. Pretreatment of cells with ryanodine, an inhibitor of Ca 2+-induced-Ca 2+-release, inhibited allicin-induced Ca 2+ transients, but the basal Ca 2+ levels were unaffected by ryanodine. Thus, allicin-induced Ca 2+ transients are most likely mediated through ryanodine-sensitive intracellular Ca 2+ stores.

Interleukin-1β Increases Prostaglandin E 2-Stimulated Adenosine 3′,5′-cyclic Monophosphate Production in Rabbit Pigmented Ciliary Epithelium

This study was designed to determine the effects of interleukin-1 on basal and prostaglandin E 2-stimulated adenosine 3′,5′-cyclic monophosphate production by primary and first passage cultures of non-transformed rabbit pigmented and non-pigmented ciliary epithelial cells. Confluent cultures of rabbit pigmented and non-pigmented ciliary epithelial cells were incubated for varying periods of time in serum-free medium with or without interleukin-1β, tumor necrosis factor-α, bacterial lipopolysaccharide, transforming growth factor-β2, cycloheximide, indomethacin and combinations of these agents. Cells were then preincubated for 10min with serum-free medium plus the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine and then stimulated for 10min with serum-free medium plus 3-isobutyl-1-methylxanthine (for basal adenosine 3′,5′-cyclic monophosphate production) or serum-free medium containing several concentrations of prostaglandin E 2and 3-isobutyl-1-methylxanthine. In certain experiments isoproterenol, vasoactive intestinal peptide, or forskolin was substituted for prostaglandin E 2. Adenosine 3′,5′-cyclic monophosphate was then extracted into ice-cold absolute ethanol and measured by radioimmunoassay. Prostaglandin E 2stimulated adenosine 3′,5′-cyclic monophosphate production in pigmented and non-pigmented ciliary epithelial cells in a dose-dependent manner. Incubation with interleukin-1β (150Uml -1) increased prostaglandin E 2-stimulated, but not basal adenosine 3′,5′-cyclic monophosphate production in pigmented ciliary epithelial cells. This interleukin-1β-induced enhancement of prostaglandin E 2-stimulated adenosine 3′,5′-cyclic monophosphate production, called the interleukin-1 effect, was not seen with non-pigmented ciliary epithelial cells. The interleukin-1 effect was dependent upon interleukin-1β concentration, time and de novo protein synthesis. The interleukin 1 effect could not be reproduced by replacing interleukin-1β with tumor necrosis factor-α or bacterial lipopolysaccharide and was specific for prostaglandin E 2, since interleukin-1β did not enhance isoproterenol-, vasoactive intestinal peptide-, or forskolin-induced adenosine 3′,5′-cyclic monophosphate production. Chronic exposure to prostaglandin E 2(during the 3hr incubation period), with or without interleukin-1β in the incubation medium, reduced subsequent prostaglandin E 2-stimulated adenosine 3′,5′-cyclic monophosphate production. Inhibition of de novo prostaglandin synthesis with indomethacin increased the interleukin-1 effect. The interleukin-1 effect was inhibited by the immunosuppressive cytokine, transforming growth factor-β2, in a dose-dependent manner. This is the first report of prostaglandin E 2-induced stimulation of adenosine 3′,5′-cyclic monophosphate production by pigmented ciliary epithelial cells and of the unique ability of interleukin-1 to increase this effect. The results are consistent with interleukin-1-induced upregulation of prostaglandin E receptors. Since transforming growth factor-β2 inhibited this interleukin-1 effect, this immunosuppressive cytokine may exert negative feedback and thus regulate the physiological consequences of the interleukin-1 effect.