Studies on regulation of the ascorbic acid transporter in a cell line derived from rabbit non-pigmented ciliary epithelium.

A cell line was derived from rabbit non-pigmented ciliary epithelium. The non-pigmented ciliary epithelium is one of the two cell layers which secrete aqueous humor into the eye and concentrate ascorbic acid in the newly-formed fluid. The cultured non-pigmented epithelial cells accumulated ascorbic acid at a rate of 3-5 pmol/micrograms protein per h. As in freshly-isolated native tissue, the ascorbate uptake mechanism was sodium-dependent and could be inhibited by phloretin (apparent Ki = 2-10(-5) M). Phorbol 12,13-dibutyrate (PDBu), a protein kinase C activator, reduced the ascorbate uptake rate. The PDBu effect was concentration-dependent; at a concentration of 10(-6) M, PDBu reduced the ascorbate uptake rate to 65% of the control value. PDBu reduced the maximal rate of ascorbate uptake (determined at 200-500 microM external ascorbate) but caused no detectable change in the Km for ascorbic acid (approx. 80 microM). The PDBu-induced inhibition of ascorbate uptake persisted in the presence of ouabain and in low sodium (25 mM Na) medium, suggesting that the effect is not secondary to a change in the sodium gradient. Furthermore, no detectable elevation of cell sodium content was seen in cells equilibrated with 22Na prior to PDBu treatment. The PDBu-induced inhibition of ascorbate uptake was apparently mediated by protein kinase C because the effect was not observed in the presence of staurosporine (10(-6) M), a protein kinase C inhibitor, or in cells in which protein kinase C was downregulated. These observations suggest that activation of protein kinase C causes inhibition of the ascorbate transporter in this cultured cell line.

Nucleotide sequence of a cDNA for the beta 2 subunit isoform of Na+,K(+)-ATPase from human retina.

Using as probe the entire human liver cDNA clone coding for the beta 2 subunit isoform of the Na+,K(+)-ATPase, which lacks the initiation codon ATG, and the entire 5'-untranslated region (Martin-Vasallo, P., Dackowski, W., Emanuel, J.R. and Levenson, R. (1989) J. Biol. Chem. 264, 4613-4618), we isolated a larger clone from a directional human adult retina cDNA library (Swaroop, A. and Xu, J. (1993) Cytogenet. Cell Genet. 64, 292-294). This clone, pNH beta 2, shows 100% homology with the nucleotide sequence of the human liver cDNA clone and also contains additional 407 nucleotides in the 5'-untranslated region, the initiation codon and a poly(A) tail. Northern blot hybridization analysis reveals that the human mRNA (3.6 kb) is approx. 300 nucleotides larger than the major transcript size expressed in rat (3.3 kb). The larger human size mRNA for the human beta 2 Na+,K(+)-ATPase indicates species differences in gene processing.

Differential gene expression in the human ciliary epithelium.

The generation of expression and subtractive libraries from the ocular ciliary body and cultured ciliary epithelial cells has been instrumental in the cloning, identification and characterization of many genes which, overall reflect a representative profile of transcripts expressed in ciliary nonpigmented, ciliary pigmented and ciliary muscle cells. The cell-specific expression of some of these genes (i.e. a neurotrophic factor, a gene associated with juvenile open glaucoma, and a visual component) reveal a degree of cell differentiation with a diversity of functions and properties higher than previously thought. The protection from light-induced oxidative reactions, free radicals and detoxification, may be partially attributed to the high level of expression in the ciliary epithelium of antioxidative enzymes (i.e., glutathione S-transferase, glutathione peroxidases, selenoprotein-P). The expression of genes encoding plasma proteins (i.e., complement component C4, alpha2-macroglobulin, apolipoprotein D) is in contrast with the view that plasma proteins in aqueous humor are synthesized outside the eye (i.e., liver). The identification of neuropeptide-processing enzymes (i.e., prohormone convertases, carboxypeptidase E, peptidyl-glycine-alpha-amidating monoxigenase), neuropeptides (i.e., secretogranin II, neurotensin) and regulatory peptides (i.e., atrial natriuretic peptide and angiotensinogen) with hypertensive and hypotensive activities provide the molecular basis to support the view that the ciliary epithelium is a neuroepithelium with neuroendocrine functions. We propose a working model to demonstrate that aqueous humor and intraocular pressure are under neuroendocrine control through regulatory peptides synthesized and released by the ciliary epithelium and targeting the peptide producing cells at the inflow system by an autocrine mechanism and/or cells at the outflow system (i.e., trabecular meshwork cells) by a paracrine mechanism. Finally, we hypothesize that these mechanisms could be entrained in the light-dark cycle following the circadian rhythm of aqueous humor and intraocular pressure.

Expression of cell surface transmembrane carbonic anhydrase genes CA9 and CA12 in the human eye: overexpression of CA12 (CAXII) in glaucoma.

PURPOSE: Carbonic anhydrase enzymes (CAs) are universally involved in many fundamental physiological processes, including acid base regulation and fluid formation and movement. In glaucoma patients, CA inhibitors are very effective in lowering intraocular pressure by reducing the rate of aqueous humour secretion mediated by the CAs in the ciliary epithelium. In this work, we investigated the expression and tissue distribution of two recently discovered CA genes CA9 (CAIX) and CA12 (CAXII) in fetal, neonatal, and adult human eyes with and without glaucoma. METHODS: CAIX and CAXII expression in 16 normal and 10 glaucomatous eyes, and in cultured non-pigmented ciliary epithelial cells (NPE) from normal and glaucoma eye donors was assessed by immunostaining. In addition, northern blot hybridisation was performed to assess expression of CA4, CA9, and CA12 mRNA in cultured NPE cells from normal and glaucoma donors. RESULTS: CAXII was localised primarily to the NPE with its expression prominent during embryonic eye development but which decreased significantly in adults. CAIX expression in the NPE was very low. The epithelium of cornea and lens occasionally expressed both enzymes at low levels during development and in adult eye, and no expression was detected in the retina. The NPE from glaucoma eyes expressed higher levels of CAXII, but not CAIX, in comparison with normal eyes. This expression pattern was retained in cultured NPE cell lines. NPE cells from a glaucoma patient showed a five-fold increase in the CA12 mRNA level with no detectable expression of CA9 mRNA. Also, no expression of the CA4 gene encoding a GPI anchored plasma membrane protein was detected on these northern blots. CONCLUSIONS: Transmembrane CAIX and CAXII enzymes are expressed in the ciliary cells and, thus, may be involved in aqueous humour production. CA12 may be a targeted gene in glaucoma.

Identification, expression and chromosome localization of a human gene encoding a novel protein with similarity to the pilB family of transcriptional factors (pilin) and to bacterial peptide methionine sulfoxide reductases.

Here we report the isolation, characterization and chromosome localization of a subtracted cDNA (CBS-1) isolated from the human ocular ciliary body which encodes a novel protein. As is deduced from the nucleotide sequence of the cDNA, CBS-1 contains an open reading frame consisting of 182 amino acids, with a molecular weight of 19.5kDa. CBS-1 shares significant nucleotide and amino acid sequence identities (residues 51 to 182) with a hypothetical 15.5kDa protein in the ANSA-GAP intergenic region (yeaA) of Escherichia coli, and the carboxyl terminal region of pilB, a transcription factor involved in the regulation of expression of pili, from Neisseria gonorrhoeae. Interestingly, CBS-1 also shares significant identity with the carboxyl terminus of the peptide-methionine sulfoxide reductase (MsrA), a repair enzyme, from Helicobacter pylori and Streptococcus pneumoniae. However, the amino terminal of CBS-1 (residues 23 to 43), which lacks homology to the amino terminal region of gonococcal pilB or pneumococcal MsrA, exhibits significant identity in a stretch of 20 amino acids, with glycine-rich proteins. By Northern blot, CBS-1, hybridized to a 0.6 to 0.7kb transcript in size, is expressed ubiquitously in many tissues, but most abundantly in the retina and ocular ciliary body, skeletal muscle and heart. An epitope-directed antibody to an amino acid sequence at the carboxyl terminus of CBS-1 recognized a main protein of 19.5kDa in ocular ciliary body extracts, and a 23kDa protein in total extracts from E. coli MC1061 cells, which expresses high levels of MsrA. The CBS-1 gene was mapped to human chromosome 10p12 between markers WI-8535 and WI-4724, and is tightly linked to the two STRP markers of D10S1789 and D10S550. We suggest that the CBS-1 gene encodes a mammalian transcription factor related to the bacterial pilB and certain bacterial MsrA homologues.

Cloning and characterization of subtracted cDNAs from a human ciliary body library encoding TIGR, a protein involved in juvenile open angle glaucoma with homology to myosin and olfactomedin.

A group of cDNAs isolated from a subtractive ciliary body library of a normal human eye donor revealed 100% identity with TIGR a candidate gene responsible for juvenile open zangle glaucoma [Science 275 (1997) 668-670]. Several structural features of the deduced human protein have been noted: a cleavable N-terminal signal peptide, a periodic repetition at the N-terminus of leucine and arginine residues at every seventh and eleven position respectively in helix conformation (leucine zipper-like motif) exhibiting homology with myosin, and with olfactomedin in the C-terminus. The mRNA for TIGR is abundantly expressed in the ciliary body, iris, heart and skeletal muscle.

Receptor-mediated phosphoinositide hydrolysis in human ocular ciliary epithelial cells.

The hydrolysis of phosphoinositides (PI) in peripheral tissues can be stimulated by a number of putative neurotransmitters and this stimulation can be blocked by specific antagonists. Epithelial cells derived from the nonpigmented layer of the ocular ciliary epithelium were transfected by simian virus 40 and grown in culture to semiconfluency. The cells were incubated in 3 microCi/ml of (3H)-myoinositol for 2 days. The accumulation of inositol phosphates in response to several agonists (carbachol, 1 mM; ATP, 100 microM; arginine vasopressin, 1 microM; and phenylephrine, 100 microM) was determined for times ranging from 5 sec to 15 min. In the presence of 10 mM LiCl, the maximum net production of the (3H)-inositol phosphates (expressed as a percent of conversion of (3H)-phospholipids) was approximately 7.5% for inositol-1 phosphate, 0.5% for inositol-1,4 bisphosphate, and 1% for inositol-1,4,5 trisphosphate. Carbachol elicited PI hydrolysis with an EC50 value of 39 +/- 9 microM. The EC50 values obtained for arginine vasopressin and ATP-initiated PI breakdown were 32 +/- 10 nM and 11.9 +/- 1 microM, respectively. Phenylephrine alone failed to stimulate the production of (3H)-inositol phosphates in these cells. The production of all (3H)-inositol phosphates in response to carbachol (1 mM) was inhibited by atropine (Ki = 0.3 nM) and the selective muscarinic antagonists 4-DAMP (Ki = 4.2 nM), pirenzepine (Ki = 102 nM) and AFDX-116 (Ki = 1.49 microM). Thus the muscarinic receptors that are coupled to PI hydrolysis in these cells have the pharmacologic characteristics of the M3 subtype.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of cDNA clones encoding the 80-kd subunit protein of the human autoantigen Ku (p70/p80) by antisera raised against ciliary processes of human eye donors.

PURPOSE: To isolate and characterize human nonpigmented ocular ciliary epithelial cDNA clones by screening a cDNA library constructed from the established human nonpigmented ciliary epithelial cell line (ODM-2) with sera raised against ciliary processes of human eye donors (cadavers). METHODS: An ODM-2 cDNA expression library in lambda Uni-ZAP-XR vector was screened with sera of mouse anti-human ciliary processes. The cDNA inserts from plaque-purified clones were sequenced by the dideoxy-chain termination method with T3 and T7 polymerase. Sera were assayed by Western blot analysis and indirect immunofluorescence and were then compared with sera, enriched in anti-Ku antibodies, collected from patients with scleroderma polymyositis overlap syndrome. RESULTS: Immunoscreening of 2.5 x 10(5) independent clones with sera induced experimentally in total tissue homogenates of human ciliary processes led to the isolation of four cDNA-positive clones. Three clones, designated A1, D1, and D3 and containing inserts measuring 1.4 kb to 1.8 kb, were found to be identical in DNA sequence analysis to that of the 80-kd subunit protein of the human autoantigen Ku (p70/p80). The fourth clone, designated D2, failed to show any significantly similarity with previously known genes. Cell extracts from human ciliary processes and ODM-2 cells were analyzed by immunoblotting with anti-human ciliary process sera. The pattern of proteins immunodetected was compared with the profile of proteins immunodetected with human anti-Ku sera from a patient with scleroderma polymyositis overlap syndrome. These results demonstrated that sera from anti-human ciliary processes recognize a variety of antigens in ODM-2 cells and are strikingly rich in antibodies to the 80-kd subunit protein of Ku (p70/p80). Furthermore, indirect immunofluorescence analysis supported the observation that anti-human ciliary processes sera labeled nuclear antigens in a human ciliary epithelium cell line and cross species in bovine ciliary epithelial cell, thus demonstrating the nuclear localization of Ku antigen. The mRNA in the human eye was found to be widely expressed in all tested ocular tissues and in ODM-2 cells. CONCLUSIONS: The ocular ciliary epithelium induces a species-specific immune response in xenogenic animals. In particular, the human ciliary epithelium elicits a strong immune recognition of the 80-kd subunit protein of the human autoantigen Ku (p70/p80) in ODM-2 cells.

Electrical membrane properties of a cell clone derived from human nonpigmented ciliary epithelium.

Intracellular potentials were measured in a SV-40 virus-transformed cell clone derived from human nonpigmented ciliary epithelium using the microelectrode technique. (1) Membrane potential averaged -50.2 mV (+/- 0.6, n = 207). (2) Increasing the extracellular K+ concentration depolarized the membrane voltage. The amplitude of this potential response was reduced in the presence of 1 mM Ba2+. (3) Superfusing the cells with a Ca2+-free solution containing 1 mM EGTA depolarized the intracellular potential and diminished the voltage response upon increasing extracellular K+. (4) Extracellular alkalinization hyperpolarized the membrane potential and increased the voltage amplitude on increasing extracellular K+. (5) Addition of ouabain immediately reduced the intracellular potential. Removing extracellular K+ depolarized membrane voltage, readdition of K+ after K+ depletion transiently hyperpolarized intracellular voltage. Both potential responses were inhibited in the presence of ouabain. (6) Replacing extracellular Cl- by cyclamate resulted in a transient depolarization followed by a hyperpolarization. In the presence of SITS or DIDS (greater than or equal to 0.1 mM) the electrical responses of the cell membrane to Cl- replacement were blocked. We conclude that cultured human nonpigmented ciliary epithelial cells possess an electrogenic Na+/K+-ATPase, a K+ conductance modulated by Ca2+ and pH, and a Cl- conductance sensitive to stilbene derivatives.

Gene expression of the neurotrophic pigment epithelium-derived factor in the human ciliary epithelium. Synthesis and secretion into the aqueous humor.

PURPOSE: To study the expression of the neurotrophic pigment epithelium-derived factor (PEDF), a protein with neurotrophic and neuronal-survival activities, by the human ocular ciliary epithelium. METHODS: Total RNA extracted from human and bovine ocular tissues were screened by Northern blot analysis with cDNA probes for PEDF. Antibodies to PEDF were used to monitor its synthesis and secretion by metabolically labeling ciliary processes in vitro with 35S-methionine, followed by immunoprecipitation. Pigment epithelium-derived factor antibodies also were used to visualize the cellular distribution of PEDF along the human and bovine ciliary epithelium. Polymerase chain reaction (PCR) and reverse transcription (RT)-PCR was used to screen cDNA libraries of tissue and cell lines derived from the ciliary epithelium to demonstrate PEDF expression. RESULTS: From a subtractive library of the human ocular ciliary body, the authors identified a cDNA clone exhibiting nucleotide homology with the PEDF. Northern blot analysis indicated that PEDF transcripts are present in all the ocular tissues in the human eye; in the bovine eye, it is expressed preferentially in the retinal pigment epithelium. RT-PCR and PCR demonstrated that the PEDF gene is still transcriptionally active in cultured cell lines derived from the bilayer of the ciliary epithelium. Immunoprecipitation and Western blot (immunoblot) analyses with antisera to the PEDF protein demonstrated that a predominant PEDF form of 46 kDa is synthesized in the ciliary body and is secreted as a glycoprotein of 50 kDa. By indirect immunofluorescence and immunocytochemistry, PEDF antibodies decorated both cell types that comprise the ciliary epithelium (nonpigmented and pigmented) and, more distinctively, the plasma-membrane domain of nonpigmented cells in the pars plicata region. CONCLUSIONS: These results reveal a new site of synthesis (ciliary epithelium) and accumulation (aqueous humor) of PEDF, and they emphasize its potential importance as a trophic factor in the neuro-differentiated functions of the human ciliary epithelium.

Pathways signaling the regulatory volume decrease of cultured nonpigmented ciliary epithelial cells.

PURPOSE: The authors identify the signaling pathways for the regulatory volume decrease (RVD) of nonpigmented ciliary epithelial (NPE) cells. The RVD is a regulatory response triggered by swelling and reflecting KCl release by NPE cells. METHODS: The cell volumes of human nonpigmented ciliary epithelial cells were measured in suspension by electronic cell sorting. Measurements were conducted in test solutions of constant ionic strength, but osmolality was varied by sucrose. RESULTS: Cyclic AMP (cAMP), forskolin, PGE2, the PKC-inhibitor staurosporine, and increasing cytoplasmic Ca2+ activity with thapsigargin all enhanced the RVD. Leukotrienes A4, D4, E4, and the protein phosphatase inhibitor okadaic acid had no detectable effect under the current experimental conditions. The cyclooxygenase inhibitor indomethacin, the epoxygenase inhibitors ketoconazole and SKF 525A, and the PKC activator DiC8 all downregulated the RVD. The addition of the cation ionophore, gramicidin, increased the RVD. In the presence of gramicidin, cAMP, PGE2, and indomethacin did not affect the RVD, but ketoconazole, DiC8, and the calcium-calmodulin blocker trifluoroperazine still inhibited--and staurosporine still enhanced--the RVD. Many of these observations are strikingly different from results reported with other cells. Anisosmotic swelling did not increase intracellular cAMP concentration. CONCLUSIONS: The pathways signaling the regulatory responses to swelling are unique for each cell type. The authors propose that hypotonic swelling of NPE cells stimulates arachidonic acid turnover, triggering PGE2-mediated upregulation of K+ channels and epoxide-mediated upregulation of Cl- channels. Swelling may also reduce endogenous PKC activity, further upregulating Cl- channels. Calcium-calmodulin plays a permissive role in upregulating the Cl- channels.

Cell-specific expression of the human Na+,K(+)-ATPase beta 2 subunit isoform in the nonpigmented ciliary epithelium.

PURPOSE: To evaluate the patterns of expression of beta subunit isoforms of the Na+,K(+)-ATPase and H+,K(+)-ATPase in the human eye and to determine the cell-specific distribution of the beta 2 subunit in the human ciliary epithelium. METHODS: Total RNA extracted from human ocular tissues was screened by Northern blot analysis with cDNA probes for the human Na+,K(+)-ATPase subunit isoforms (beta 1 and beta 2) or the H+,K(+)-ATPase (alpha and beta) subunits. Antibodies were raised to the amino and carboxyl terminal regions of the human beta 2 isoform. Polymerase chain reaction was used to verify the expression of beta 2 subunit in nonpigmented ciliary epithelial cells (NPE). RESULTS: Transcripts for the Na+,K(+)-ATPase beta 1 and beta 2 subunit isoforms were present at different levels in all the ocular tissues except the lens, which expressed only beta 1. No transcripts for the alpha or beta subunits of the H+,K(+)-ATPase were detected in the eye. Isoform beta 2 specific anti-peptide antibodies V15E (N-terminus) and A18R (C-terminus) recognized a 55- to 60-kDa protein in the ciliary epithelium and the core protein of 32 kDa after N-glycanase treatment. Immunocytochemical localization within the ciliary epithelium indicates that the Na+,K(+)-ATPase beta 2 isoform is expressed preferentially in the NPE cells. The expression of Na+,K(+)-ATPase beta 2 isoform in the human NPE cell line, ODM-2, was confirmed by polymerase chain reaction amplification and Southern blot analysis. CONCLUSIONS: The Na+,K(+)-ATPase beta 2 subunit isoform, but not H+,K(+)-ATPase, was expressed widely in ocular tissues of the human eye. The restricted cellular distribution of beta 2 isoform within the NPE cells represents an important differential gene marker associated with the multiple alpha subunit isoforms of Na+,K(+)-ATPase.

Effect of acetylcholine on membrane potential of cultured human nonpigmented ciliary epithelial cells.

Human nonpigmented ciliary epithelial cells (NPE) were grown in tissue culture after transformation with an origin-defective mutant of SV-40 DNA. In these cells membrane potentials (V) were measured using the microelectrode technique. Addition of 10(-4) M acetylcholine led to a bisphasic voltage response. An immediate, transient hyperpolarization was followed by a sustained depolarization below the steady state level. These responses were irreversibly blocked by 10(-5) M atropine. In Ca2+-free media the initial addition of acetylcholine resulted in an unchanged voltage response. A second application of acetylcholine in Ca2+-free solution evoked only an abortive response of V, and further addition had no effect on V. In the presence of Ca2+ channel blockers (10(-5) M verapamil, 1 mM Co2+) the acetylcholine-induced response of the membrane potential was not changed. The initial hyperpolarization induced by acetylcholine was reduced by 33 +/- 3% (n = 6) in the presence of 2 mM Ba2+ and by 79 +/- 6% (n = 6) in the presence of 1 mM quinidine. Moreover, the amplitude of the hyperpolarization was dependent on the extracellular K+ concentration. With increasing extracellular K+ concentration (and decreasing transmembrane K+ gradient) the acetylcholine-induced hyperpolarization was reduced. To further elucidate the role of Ca2+ in the acetylcholine-induced responses, we measured cytoplasmic Ca2+ activity using the fluorescence of intracellularly trapped Fura-2. Cytoplasmic Ca2+ activity increased immediately and transiently upon addition of acetylcholine. We conclude that acetylcholine transiently hyperpolarizes V in cultured human NPE by activation of K+ channels mediated by mobilization of Ca2+ from intracellular stores.

Crocin, safranal and picrocrocin from saffron (Crocus sativus L.) inhibit the growth of human cancer cells in vitro.

Extracts of saffron (Crocus sativus L.) have been reported to inhibit cell growth of human tumor cells. In order to study the cytotoxic effect of the characteristic compounds of saffron spice, we have isolated crocin, crocetin, picrocrocin and safranal. Doses inducing 50% cell growth inhibition (LD50) on HeLa cells were 2.3 mg/ml for an ethanolic extract of saffron dry stigmas, 3 mM for crocin, 0.8 mM for safranal and 3 mM for picrocrocin. Crocetin did not show cytotoxic effect. Cells treated with crocin exhibited wide cytoplasmic vacuole-like areas, reduced cytoplasm, cell shrinkage and pyknotic nuclei, suggesting apoptosis induction. Considering its water-solubility and high inhibitory growth effect, crocin is the more promising saffron compound to be assayed as a cancer therapeutic agent.

Isolation and characterization of cell-specific cDNA clones from a subtractive library of the ocular ciliary body of a single normal human donor: transcription and synthesis of plasma proteins.

A subtractive cDNA library was developed for the purpose of identifying cell-specific genes expressed within the human ocular ciliary body, a tissue responsible for regulating aqueous humor secretion and intraocular pressure. Partial DNA sequence of a large number of cDNA clones and homology searches of nucleic acid and protein databases revealed significant homologies to at least 90 independently known genes. A group of biologically significant genes that were previously not known to have transcriptional expression in the ciliary body, complement component C4; alpha 2 macroglobulin; selenoprotein-P; and apolipoprotein D, were further demonstrated by Northern hybridization. Antibodies to these and other proteins (i.e., tyrosinase-related protein and pigment epithelium-derived factor) confirmed their cell-restricted expression in ciliary epithelial cells (pigmented, nonpigmented), or vascular endothelial cells. We provide evidence that two human plasma proteins, complement component C4 and alpha 2-macroglobulin are metabolically labeled with [35S]methionine in ciliary processes explants, suggesting that the ciliary body is an organ of synthesis and secretion of plasma proteins present in aqueous humor. These results challenge the notion that plasma proteins in aqueous humor are imported from outside of the eye. The subtractive cDNA library reported in this work should be very useful for identifying potential candidate genes in ocular abnormalities affecting the ciliary body, or involved in the regulation of intraocular pressure.

Cloning of the bovine plasma selenium-dependent glutathione peroxidase (GP) cDNA from the ocular ciliary epithelium: expression of the plasma and cellular forms within the mammalian eye.

In the anterior segment of the mammalian eye, the ocular ciliary epithelium produces the aqueous humor, a fluid that nourishes and protects the avascular tissues from oxidative stress. This report details the results of a study of molecular cloning, sequencing, and expression of plasma glutathione peroxidase (GPx-P) from the bovine ocular ciliary epithelium. The bovine GPx-P cDNA contains an open reading frame of 226 amino acids with a calculated molecular weight of 24,860. The corresponding amino acid sequence showed an overall identity of 88% with the human GPx-P, 88.5% with the rat GPx-P, and 46.4% with the cellular bovine glutathione peroxidase (GPx-1). The levels of GPx-P and GPx-1 transcripts in ocular tissues were analyzed and the ciliary epithelium was found to express the highest levels of GPx-P transcripts in human and bovine eyes, whereas the cornea of calf eyes expressed the highest levels of GPx-1 transcripts. Surprisingly, the lens, on which oxidants have profound effects leading to cataract formation, expressed the lowest levels of GPx-P and GPx-1 transcripts in human donor eyes. These results provide new evidence of differential gene expression of the GPx-P and GPx-1 forms in the mammalian eye and stresses the functional role of the ocular ciliary epithelium in protecting the anterior segment of the eye from oxidative damage.

Molecular cloning of the bovine CD9 antigen from ocular ciliary epithelial cells.

The ciliary epithelium is a bilayer of epithelial cells responsible for the formation and secretion of aqueous humor in the mammalian eye. We have isolated a cDNA clone from a lambda gt11 cDNA library of bovine ocular ciliary epithelial cells encoding the CD9 antigen, a member of a new family of transmembrane proteins. The bovine CD9 clone contains an open reading frame of 226 amino acids (M(r) 24,860). The deduced amino acid sequence from the bovine CD9 cDNA clone shows 83.5% identity with the human counterpart isolated from megakaryocytes, and a lower degree of identity with a group of related antigens (TAPA-1, C0-029, CD53, MRC OX-44, ME491, CD63, CD37, and Sm23) involved in growth regulation. Analysis of bovine ocular tissues reveals that the CD9 gene encodes a 1.4 kb mRNA which is detectable predominantly in cornea and at low levels in ciliary epithelium, retina, iris, and lens. Normal and transformed cell lines established from the ocular ciliary epithelium exhibited significant levels of CD9 transcripts. These results raise questions regarding possible roles of CD9 in the anterior segment of the eye.

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.

Inhibition of 11beta-hydroxysteroid dehydrogenase type 1 lowers intraocular pressure in patients with ocular hypertension.

BACKGROUND: Intraocular pressure (IOP) is maintained by a balance between aqueous humour (AH) production (dependent on sodium transport across a ciliary epithelial bi-layer) and drainage (predominantly through the trabecular meshwork). In peripheral epithelial tissues, sodium and water transport is regulated by corticosteroids and the 11beta-hydroxysteroid dehydrogenase (11beta-HSD) isozymes (11beta-HSD1 activating cortisol from cortisone, 11beta-HSD2 inactivating cortisol to cortisone). AIM: To analyse expression of 11beta-HSD in the human eye and investigate its putative role in AH formation. DESIGN: Multipart prospective study, including a randomized controlled clinical trial. METHODS: The expression of 11beta-HSD1 in normal human anterior segments was evaluated by in situ hybridization (ISH). RT-PCR for 11beta-HSDs, glucocorticoid and mineralocorticoid receptors (GR, MR) was performed on human ciliary body tissue. AH cortisol and cortisone concentrations were measured by radioimmunoassay on specimens taken from patients with primary open-angle glaucoma (POAG) and age-matched controls. Randomized, placebo-controlled studies of healthy volunteers and patients with ocular hypertension (OHT, raised IOP but no optic neuropathy) assessed the effect of oral carbenoxolone (CBX, an inhibitor of 11beta-HSD) on IOP. RESULTS: ISH defined expression of 11beta-HSD1 in the ciliary epithelium, while RT-PCR analysis of ciliary body tissue confirmed expression of 11beta-HSD1, with additional GR and MR, but not 11beta-HSD2 expression. In both POAG patients and controls, AH concentrations of cortisol exceeded those of cortisone. The CBX-treated healthy volunteers who demonstrated the largest change in urinary cortisol metabolites, indicative of 11beta-HSD1 inhibition, had the greatest fall in IOP. Patients with OHT showed an overall reduction of IOP by 10% following CBX administration, compared to baseline (p<0.0001). DISCUSSION: CBX lowers IOP in patients with ocular hypertension. Our data suggest that this is mediated through inhibition of 11beta-HSD1 in the ciliary epithelium. Selective and topical inhibitors of 11beta-HSD1 could provide a novel treatment for patients with glaucoma.

Expression of the TIGR gene in the iris, ciliary body, and trabecular meshwork of the human eye.

Mutations in the the glaucoma gene GCL1A, also known as trabecular meshwork glucocorticoid response (TIGR) or myocilin (Myoc), have been shown to be associated with juvenile-onset primary open-angle glaucoma. Very little is known about the pattern of expression of the TIGR gene in human ocular tissues. In-situ hybridization experiments demonstrated the localization of TIGR mRNA in cells throughout the iris, ciliary muscle, and the filtering portion of the trabecular meshwork of normal eye donors. The expression of TIGR protein was investigated by Western blot using an epitope-directed antibody to the carboxy terminus region of TIGR. This antibody was able to distinguish a recombinant TIGR fusion protein from a truncated TIGR form containing the naturally occurring Gln(368)-->stop mutation. In tissue extracts from the iris, ciliary body, and trabecular meshwork, the antibody recognized a major protein band of 57-kDa molecular mass. Deglycosylation treatment with PNGase F, NANase II, and O-glycosidase indicated that the 57-kDa protein in these tissues was unglycosylated. In agreement with this observation, in coupled in-vitro transcription/translation systems, the 57-kDa TIGR protein was unaffected by the presence of the processing and glycosylation activities of canine pancreatic microsomal membranes. These findings support the view that the expression of TIGR mRNA in cells of the iris, ciliary body, and trabecular meshwork correlates with that of TIGR protein, and that the 57-kDa TIGR protein was unglycosylated. These results, which are in contrast with earlier reports, raise the possibility that the TIGR protein might be processed into distinct forms in a tissue-specific manner.