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Aims: The aim of the present study was to immunohistochemically investigate the expression and prognostic significance of putative cancer stem cell markers CD117 (c-kit), CD34, CD20 and CD15 in a cohort of patients with primary choroidal and ciliary body melanoma., Methods: The immunohistochemical expression of these markers was evaluated using 3,3'-diaminobenzidine tetrahydrochloride (DAB) and 3-amino-9-ethylcarbazole (AEC) chromogens on paraffin-embedded tissue samples from 40 patients who underwent enucleation in the period from 1985 through 2000. Thirty-one patients had adequate tissue specimens for the analysis., Results: CD117 overexpression was observed in 12 of the 31 samples (39%) when AEC chromogen was used and in 14 of 26 (54%) samples when DAB was used. CD15 positivity was seen in three out of 30 (10%) samples with AEC and in six out of 26 (23%) samples with DAB. CD20 and CD34 exhibited no positivity in the tested samples. During the average follow-up time of 8.7 years (range 0.5-22 years), 17 patients (55%) died due to metastatic disease. The Kaplan-Meier plots showed a significantly shorter overall and disease-free survival in CD117-positive patients when the AEC chromogen was used. CD15 expression was not associated with patients' survival. In multivariate analysis, patients expressing the CD117 AEC had 4.13 times higher risk of lethal outcome in comparison with CD117 AEC negative patients., Conclusions: Our retrospective cohort study has for the first time demonstrated a small proportion of CD15-positive uveal melanomas. CD117 AEC overexpression was associated with a worse outcome in patients with choroidal and ciliary body melanoma. Further studies should confirm the validity of these observations and their potential for targeted treatment modalities., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Glutaredoxin2 (Grx2) is a mitochondrial isozyme of the cytosolic glutaredoxin1 (thioltransferase or TTase). Both belong to the large oxidoreductase family and play an important role in maintaining thiol/disulfide redox homeostasis in the cells. Grx2 is recently found in the lens where its activities of disulfide reductase and peroxidase, similar to TTase, can protect the lens against oxidative stress. Since other eye tissues are also highly sensitive to oxidative stress, and TTase's distribution in the eye is known, we focused on this study by investigating the Grx2 distribution in the ocular tissues in comparison to the lens. Fresh porcine eyes were dissected into cornea, iris, ciliary body, the lens, vitreous humor, retina, and optic nerve. Each tissue (pooled from three eyes) was homogenized and processed for mitochondrial isolation. The mitochondrial fraction was analyzed for Grx2 protein using Western blotting with anti-Grx2 antibody, and Grx2 activity using the published procedure. The eye tissues were also measured for Grx2 mRNA expression by RT-PCR with GAPDH as the control. Grx2-rich mouse liver and purified recombinant mouse Grx2 were used as positive controls for the above analyses. It was found that Grx2 was present in all the tested ocular tissues, except vitreous humor. In comparison with the mouse liver, the protein levels of Grx2 in porcine ciliary body and the lens were 27-fold and 0.75-fold, respectively. Comparing to the lens, Grx2 protein was highest in the ciliary body (13.5-fold), followed by retina (9.2-fold), iris and optic nerve (2-fold), and cornea (1.2-fold). Enzyme activity assays showed that the retina had the highest Grx2 specific activity (3.9 mU/mg protein), followed by ciliary body (3.1 mU/mg), the lens (0.58 mU/mg), and optic nerve (0.32 mU/mg). Grx2 gene expression in these ocular tissues was further confirmed by RT-PCR analysis. Grx2 mRNA expression showed the highest in ciliary body, followed by retina, optic nerve, cornea, iris, and the lens. No Grx2 mRNA, protein or enzyme activity could be found in the vitreous humor. The results indicate that Grx2 level was higher in eye tissues rich in vasculature and mitochondria (i.e. ciliary body and retina), corroborating with the levels of mRNA expression and Grx2 activity. The rich presence of Grx2 in these tissues is also consistent with their known sensitivity to oxidative stress., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The nitric oxide (NO) donor sodium nitroprusside (SNP) is known to reduce aqueous humor (AH) secretion in the isolated porcine eye. Previously, SNP was found to inhibit Na,K-ATPase activity in nonpigmented ciliary epithelium (NPE), AH-secreting cells, through a cGMP/protein kinase G (PKG)-mediated pathway. Here we show Src family kinase (SFK) activation in the Na,K-ATPase activity response to SNP. Ouabain-sensitive (86) Rb uptake was reduced by >35% in cultured NPE cells exposed to SNP (100 µM) or exogenously added cGMP (8-Br-cGMP) (100 µM) and the SFK inhibitor PP2 (10 µM) prevented the response. Ouabain-sensitive ATP hydrolysis was reduced by ~40% in samples detected in material obtained from SNP- and 8-Br-cGMP-treated cells following homogenization, pointing to an intrinsic change of Na,K-ATPase activity. Tyrosine-10 phosphorylation of Na,K-ATPase α1 subunit was detected in SNP and L-arginine-treated cells and the response prevented by PP2. SNP elicited an increase in cell cGMP. Cells exposed to 8-Br-cGMP displayed SFK activation (phosphorylation) and inhibition of both ouabain-sensitive (86) Rb uptake and Na,K-ATPase activity that was prevented by PP2. SFK activation, which also occurred in SNP-treated cells, was suppressed by inhibitors of soluble guanylate cyclase (ODQ; 10 µM) and PKG (KT5823; 1 µM). SNP and 8-Br-cGMP also increased phosphorylation of ERK1/2 and p38 MAPK and the response prevented by PP2. However, U0126 did not prevent SNP or 8-Br-cGMP-induced inhibition of Na,K-ATPase activity. Taken together, the results suggest that NO activates guanylate cyclase to cause a rise in cGMP and subsequent PKG-dependent SFK activation. Inhibition of Na,K-ATPase activity depends on SFK activation., (© 2013 Wiley Periodicals, Inc.)

Purpose: The nonpigmented ciliary epithelium (NPE) is rich in soluble adenylyl cyclase (sAC), a proposed cytoplasmic bicarbonate sensor. Here, we examine the contribution of sAC to an increase in cyclic AMP (cAMP) and changes in a key ion transporter, H(+)-ATPase, in NPE exposed to acetazolamide, a carbonic anhydrase inhibitor (CAI)., Methods: Cyclic AMP was measured by radioimmunoassay in primary cultured porcine NPE. The pH-sensitive dye BCECF was used to examine cytoplasmic pH regulation. Subcellular protein translocation was examined by Western blot., Results: A transient cAMP increase, detectable within minutes of acetazolamide treatment, was prevented by KH7, a specific sAC inhibitor. Following 10-minute exposure to acetazolamide, the abundance of H(+)-ATPase B1 subunit and sAC was doubled in a plasma membrane-rich fraction, suggesting subcellular translocation. Similar evidence of H(+)-ATPase translocation was observed in NPE exposed to 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Consistent with increased capacity for proton export, acetazolamide increased the rate of pH recovery from acidification. KH7 and bafilomycin A1, an inhibitor of H(+)-ATPase, both prevented the stimulatory effect of acetazolamide on pH recovery. In a parallel study, H(+)-ATPase abundance was found to be higher in the plasma membrane of HEK293 cells that overexpress sAC compared to the normal HEK293 cells. HEK cells that overexpress sAC and had higher H(+)-ATPase abundance displayed a faster rate of pH recovery and greater sensitivity to KH7., Conclusions: Acetazolamide increases cAMP in a response that involves activation of sAC. Subcellular translocation of H(+)-ATPase and an increase in the capacity for proton export by acetazolamide-treated NPE cells is a cAMP-dependent response.

Objective: To explore the impact of combination use of prostaglandin analogue and cholinergic agonists on main matrix metalloproteinases (MMPs) synthesized by albino rabbit ciliary muscle., Methods: Normal adult albino rabbits were divided into the control group, 2% pilocarpine group, 0.004% travoprost group and travoprost plus pilocarpine group. Two rabbits in the control group were executed after treated with normal saline for one day. Two rabbits were separately executed on the 7th, 14th and 24th day of the treatment in each drug treated group. In each subgroup ciliary muscle band of 4 eyes was taken and made into homogenate. The MMPs activities of 10 subgroups were assayed by zymography. Bands' intensity which represents the activity of MMPs was measured by the UltraViolet Illumination system., Results: A bright band of MMP-1/2 was showed on each lane at the position corresponding to the molecular weight of 62 kD in the ciliary smooth muscles electrophoresis. When ion Zn and Ca was displaced by MMPs inhibitor EDTA, this bright band disappeared. Compared with the control group, MMP1/2 activity increased by 4.0%, 4.1% and 14.0% after 7, 14 and 24 days of pilocarpine treatment. Corresponding data was 23.2%, 61.7% and 111.5% in the travoprost group and 49.3%, 68.0% and 88.4% in the travoprost plus pilocarpine group., Conclusions: Pilocarpine has little effect on activity of MMP1/2. Travoprost can increase activity of MMP1/2 gradually. Activity of MMP1/2 is rapidly increased by pilocarpine combined with travoprost, but shows small change with the prolonged treatment.

We have previously shown that the eye is a mineralocorticoid-sensitive organ and we now question the role of mineralocorticoid receptor (MR) in ocular inflammation. The endotoxin-induced uveitis (EIU), a rat model of human intraocular inflammation, was induced by systemic administration of lipopolysaccharide (LPS). Evaluations were made 6 and 24 hours after intraocular injection of aldosterone (simultaneous to LPS injection). Three hours after onset of EIU, the MR and the glucocorticoid metabolizing enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression were down-regulated in iris/ciliary body and the corticosterone concentration was increased in aqueous humor, altering the normal MR/glucocorticoid receptor (GR) balance. At 24 hours, the GR expression was also decreased. In EIU, aldosterone reduced the intensity of clinical inflammation in a dose-dependent manner. The clinical benefit of aldosterone was abrogated in the presence of the MR antagonist (RU26752) and only partially with the GR antagonist (RU38486). Aldosterone reduced the release of inflammatory mediators (6 and 24 hours: TNF-α, IFN-γ, MIP-1α) in aqueous humor and the number of activated microglia/macrophages. Aldosterone partly prevented the uveitis-induced MR down-regulation. These results suggest that MR expression and activation in iris/ciliary body could protect the ocular structures against damages induced by EIU.

Background: The role played by the nitric oxide (NO) signaling pathway in the aqueous humor dynamics is still unclear. This study was designed to investigate the expression and distribution of NO synthase (NOS) isoforms and guanylate cyclase (GC) in human ciliary body, trabecular meshwork and the Schlemm's canal., Methods: Twelve eyes after corneal transplantation were used. Expression of three NOS isoforms (i.e. neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS)) and GC were assessed in 10 eyes by immunohistochemical staining using monoclonal or polyclonal antibody of NOS and GC. Ciliary bodies were dissected free and the total proteins were extracted. Western blotting was performed to confirm the protein expression of 3 NOS isoforms and GC., Results: Expression of 3 NOS isoforms and GC were observed in the ciliary epithelium, ciliary muscle, trabecular meshwork and the endothelium of the Schlemm's canal. Immunoreactivity of nNOS was detected mainly along the apical cytoplasmic junction of the non-pigmented epithelium (NPE) and pigmented epithelial (PE) cells. Protein expressions of 3 NOS isoforms and GC were confirmed in isolated human ciliary body by Western blotting., Conclusions: The expression of NOS isoforms and GC in human ciliary body suggest the possible involvement of NO and cyclic guanosine monophosphate (cyclic GMP, cGMP) signaling pathway in the ciliary body, and may play a role in both processes of aqueous humor formation and drainage.

Glaucoma is a leading cause of blindness affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure (IOP). IOP results from the resistance to drainage of aqueous humor (AH) produced by the ciliary body in a process requiring bicarbonate. Once secreted into the anterior chamber, AH drains from the eye via two pathways: uveoscleral and pressure-dependent or conventional outflow (C(t)). Modulation of "inflow" and "outflow" pathways is thought to occur via distinct, local mechanisms. Mice deficient in the bicarbonate channel bestrophin-2 (Best2), however, exhibit a lower IOP despite an increase in AH production. Best2 is expressed uniquely in nonpigmented ciliary epithelial (NPE) cells providing evidence for a bicarbonate-dependent communicative pathway linking inflow and outflow. Here, we show that bicarbonate-sensitive soluble adenylyl cyclase (sAC) is highly expressed in the ciliary body in NPE cells, but appears to be absent from drainage tissues. Pharmacologic inhibition of sAC in mice causes a significant increase in IOP due to a decrease in C(t) with no effect on inflow. In mice deficient in sAC IOP is elevated, and C(t) is decreased relative to wild-type mice. Pharmacologic inhibition of sAC did not alter IOP or C(t) in sAC-deficient mice. Based on these data we propose that the ciliary body can regulate C(t) and that sAC serves as a critical sensor of bicarbonate in the ciliary body regulating the secretion of substances into the AH that govern outflow facility independent of pressure.

Purpose: Cyclooxygenase-2 (COX-2) is an enzyme involved in neoplastic processes. The purpose of the present study is to investigate COX-2 expression in the normal human eye and the expression pattern in selected eye tumours involving COX-2 expressing cells., Methods: Immunohistochemical staining using antibodies against COX-2 was performed on paraffin sections of normal human eyes and selected eye tumours arising from cells expressing COX-2., Results: Cyclooxygenase-2 expression was found in various structures of the normal eye. Abundant expression was seen in the cornea, iris, ciliary body and retina. The COX-2 expression was less in tumours deriving from the ciliary epithelium and also in retinoblastoma., Conclusion: Cyclooxygenase-2 is constitutively expressed in normal human eyes. The expression of COX-2 is much lower in selected eye tumours involving COX-2 expressing cells., (© 2009 The Authors. Journal compilation © 2009 Acta Ophthalmol.)

Adenosine is known to exert multiple functions within the eye. The aim of this report was to find out if adenosine can be produced locally in the choroid and ciliary body. Therefore, I investigated the distribution of ecto-5'-nucleotidase (5'-NT), the key enzyme for the production of extracellular adenosine. This report provides evidence that 5'-NT is expressed in the choroid and in the ciliary body (and its processes) of the rat eye, predominantly in endothelial cells. These locations of 5'-NT indicate strategically important production sites of adenosine regulating choroid and ciliary body functions (e.g., blood flow, aqueous fluid production, and immune response)., (2010 Wiley-Liss, Inc.)

Purpose: Cytochrome P450 (CYP) enzymes metabolize endogenous compounds such as steroid hormones, fatty acids, and xenobiotics, including drugs and carcinogens. Expression of CYP enzymes in ocular tissues is poorly known. However, mutations in the CYP1B1 gene have been linked to congenital glaucoma. The aim of the present study was to investigate the expression and regulation of cytochrome P450 enzymes in a human nonpigmented ciliary epithelial cell line., Methods: Expression of mRNAs for major xenobiotic metabolizing CYPs in families 1-3 and regulatory factors involved in the induction of CYPs was studied using reverse transcriptase-polymerase chain reaction. For induction studies, the cells were treated with dexamethasone or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 hours. RNA and immunoblotting analysis were used to study CYP induction. Transcriptional regulation of CYP1B1 gene was studied by transient transfection of reporter gene constructs., Results: mRNAs of CYP1A1, CYP1B1, and CYP2D6 and of the regulatory factors aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator, and glucocorticoid receptor were expressed in the human nonpigmented ciliary epithelial cell line. CYP1B1 mRNA was strongly and dose dependently induced by TCDD. CYP1B1 protein was detected only after TCDD treatment of the human nonpigmented ciliary epithelial cells. CYP1B1 promoter was activated by TCDD. The major drug-metabolizing enzymes CYP1A2, CYP2Cs, and CYP3As were not detected in these cells, and dexamethasone treatment had no effect on CYP expression., Conclusions: TCDD potently induces CYP1B1 mRNA in human nonpigmented ciliary epithelial cells, suggesting the involvement of an AHR-mediated pathway in the regulation of ciliary CYP1B1 expression.

Purpose: An active local renin-angiotensin system (RAS) has recently been found in the human eye. The aim of the present study was to compare the activities of central RAS enzymes (ACE1 and 2) in porcine ocular tissues, morphologically and physiologically close to the human eye. In addition, the effects of three ACE-inhibitory tripeptides on these enzymes were evaluated., Methods: Enucleated fresh porcine eyes were used. Activities of ACE1 and ACE2 and their inhibition by bioactive tripeptides (Ile-Pro-Pro, Val-Pro-Pro, Leu-Pro-Pro) as well as by a standard ACE-inhibitor captopril were assayed in the vitreous body, the retina and the ciliary body using fluorometric detection methods., Results: Activity of ACE1 as well as ACE2 was found in all tissues evaluated. ACE1 activity was markedly higher in the ciliary body (3.7 +/- 0.7 mU/mg protein) than in retina (0.2 +/- 0.02 mU/mg), whereas ACE2 activities in the ciliary body (0.2 +/- 0.02 mU/mg) and retina (0.2 +/- 0.01 mU/mg) were at the same level. In the vitreous body ACE1 activity (8.2 +/- 0.31 nmol/min/mL) was manifold compared to that of ACE2 (0.1 +/- 0.02 nmol/min/mL). The tripeptides inhibited ACE1 at one-thousandth of the concentration needed to inhibit ACE2. All peptides studied evinced about equal inhibitory activities., Conclusion: To our knowledge the present findings constitute the first evidence of ACE2 activity in the ciliary and vitreous bodies, in addition to previously described activity in the retina. The known favorable effects of ACE2 products vs. those of ACE1 suggest a counterbalancing interaction of these two enzyme homologues in physiological regulation of ocular circulation and pressure and possible protective role in certain ophthalmic disorders such as glaucoma and diabetic retinopathy.

Purpose: Lysyl oxidase (LOX) cross-links the side chain of collagen and elastin and thereby contributes to extracellular matrix (ECM) integrity. ECM remodeling is seen in various ocular diseases. Until now, there have been no reports on the LOX enzyme's activity in ocular tissues. The purpose of this study was to estimate LOX activity and expression in human donor ocular tissues and to measure the specific activity of LOX in the vitreous of proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RRD)., Method: Human donor eyeballs obtained from an eye bank were used to study tissue distribution of LOX. Human vitreous specimens were obtained during vitreoretinal surgery from PDR (n = 16) and RRD (n = 10). LOX activity was estimated by N-acetyl-3,7-dihydroxyphenoxazine assay, immunohistochemistry, and real-time polymerase chain reaction (RT-PCR). Matrix metalloprotease (MMP)-2 and -9 were quantified in the vitreous by sandwich enzyme-linked immunosorbent assay (ELISA)., Results: The specific activity of LOX in ocular tissues was on the order of vitreous, iris ciliary body, lens, choroid RPE, and retina, which were comparable by mRNA expression and immunolocalization. The vitreous level of LOX activity decreased significantly in PDR and RRD, with an increase in total MMP-2 and -9 levels compared with normal donor vitreous., Conclusions: LOX activity showed a statistically significant decrease in the vitreous of PDR and RRD relative to control specimens. This effect can contribute to the inadequate collagen cross-linking that causes the ECM changes that occur in these diseases.

Background: Latanoprost, a prostaglandin F2alpha analog, has been shown to be an effective intraocular pressure lowering agent which acts by inducing ciliary muscle cells to synthesise matrix metalloproteinases. However, the response of ciliary melanocytes to latanoprost has never been reported. This research has investigated the ability of latanoprost to induce matrix metalloproteinase-1 expression in human ciliary melanocytes, and thereby advance the understanding of the mechanism of PGF(2alpha) in decreasing intraocular pressure., Methods: In vitro human ciliary melanocytes were treated for 48 hours with five different concentrations of latanoprost (100, 150, 200, 500, and 1000 nmol/L). Ciliary melanocytes treated with 0.01% ethanol (vehicle) were used as a control. The expression of matrix metalloproteinase-1 in ciliary melanocytes was determined by Western blotting and immunofluorescent staining., Results: Western blotting showed that the expression of matrix metalloproteinase-1 in ciliary melanocytes was induced by latanoprost, and the level of expression was dependent on the concentration of latanoprost in the culture medium. Immunofluorescent staining showed that matrix metalloproteinase-1 was confined to the ciliary melanocyte cytoplasm., Conclusions: Latanoprost induced the expression of matrix metalloproteinase-1 in human ciliary melanocytes in a dose-dependent manner. Ciliary melanocytes, as well as ciliary muscle cells, may also play an important role in uveoscleral outflow modulation.

Studies were designed to examine the roles of individual protein kinase C (PKC) isoforms in the prostaglandin F(2alpha) (PGF(2alpha))-induced matrix metalloproteinase-2 (MMP-2) secretion from human ciliary muscle cells. Studies utilized primary cultures of human ciliary muscle cells. Individual PKC isoforms were detected by Western blotting, using PKC-isoform-specific antibodies. To evaluate MMP-2 secretion, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 4 h and the media analyzed for MMP-2 by Western blotting. To assess ERK1/2 activation, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 5 min and cell lysates analyzed for ERK1/2 phosphorylation by Western blot analysis. To evaluate the roles of individual PKC isoforms, cells were pretreated with PKC inhibitors or siRNAs prior to the addition of PGF(2alpha). In cultured human ciliary muscle cells, the PKC isoforms exhibiting the highest level of expression were PKCalpha, epsilon, iota and lambda. The delta and eta isoforms exhibited moderate levels of expression and beta, gamma, and phi were not detected. The administration of PGF(2alpha) (1 micromol/L) primarily induced the translocation of PKCepsilon from cytosol to the membrane fraction, as well as increased MMP-2 secretion and ERK1/2 phosphorylation. The secretion of MMP-2 was inhibited by pretreatment with the broad-range PKC inhibitor, chelerythrine chloride; however, this response was not blocked by Go-6976, an inhibitor of conventional PKC isoforms. The PGF(2alpha)-induced secretion of MMP-2 was also blocked by pretreatment with the PKCepsilon-selective peptide translocation inhibitor, EAVSLKPT, or the transfection of siRNA-targeting PKCepsilon. The activation of ERK1/2 was inhibited by chelerythrine and the PKCepsilon translocation inhibitor. Human ciliary muscle cells express the alpha, epsilon, iota and lambda PKC isoforms. Stimulation of FP receptors in these cells activates PKCepsilon, resulting in ERK1/2 activation and an eventual increase in MMP-2 secretion. These data support the idea that the activation of FP receptors in vivo modulate uveoscleral outflow through the PKCepsilon-dependent secretion of MMPs.

The rate of aqueous humor formation sequentially across the pigmented (PE) and nonpigmented (NPE) ciliary epithelial cell layers may not be uniform over the epithelial surface. Because of the tissue's small size and complex geometry, this possibility cannot be readily tested by conventional techniques. Rabbit iris-ciliary bodies were divided, incubated, quick-frozen, cryosectioned, and freeze-dried for electron probe X-ray microanalysis of the elemental contents of the PE and NPE cells. We confirmed that preincubation with ouabain to block Na(+),K(+)-ATPase increases Na(+) and decreases K(+) contents far more anteriorly than posteriorly. The anterior and posterior regions were the iridial portion of the primary ciliary processes and the pars plicata, respectively. Following interruption of gap junctions with heptanol, ouabain produced smaller changes in anterior PE cells, possibly reflecting higher Na(+) or K(+) permeability of anterior NPE cells. Inhibiting Na(+) entry selectively with amiloride, benzamil, or dimethylamiloride reduced anterior effects of ouabain by approximately 50%. Regional dependence of net secretion was also assessed with hypotonic stress, which stimulates ciliary epithelial cell regulatory volume decrease (RVD) and net Cl(-) secretion. In contrast to ouabain's actions, the RVD was far more marked posteriorly than anteriorly. These results suggest that 1) enhanced Na(+) reabsorption anteriorly, likely through Na(+) channels and Na(+)/H(+) exchange, mediates the regional dependence of ouabain's actions; and 2) secretion may proceed primarily posteriorly, with secondary processing and reabsorption anteriorly. Stimulation of anterior reabsorption might provide a novel strategy for reducing net secretion.

Purpose: To investigate the mRNA and protein expression of nitric oxide synthase (NOS) in human ciliary bodies in vitro. The effect of the adenylcyclase activator forskolin and/or the alpha2-adrenergic agonist brimonidine (an ocular hypotensive agent that inhibits aqueous humor formation) on NOS mRNA or protein expression was also studied., Methods: Frozen human ciliary bodies obtained from local eye bank were thawed and incubated with 0.1 mM forskolin for 24 h in the absence or in the presence of 100 muM brimonidine. The mRNA and protein expression of three NOS isoforms (neuronal NOS or nNOS, inducible NOS or iNOS, endothelial NOS or eNOS) were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively., Results: mRNA and protein expression of three NOS isoforms were detected in human ciliary bodies. Forskolin significantly up-regulated the mRNA and protein expression of nNOS, but not that of iNOS or of eNOS. In the presence of brimonidine, the forskolin-induced up-regulation of nNOS mRNA or protein expression was significantly inhibited., Conclusions: In human ciliary body (where aqueous humor is produced), brimonidine inhibits the up-regulation of nNOS expression induced by forskolin.

Increasing biological findings argue for the importance of cholesterol-24S-hydroxylase (CYP46A1) in cholesterol homeostasis in cerebral structures. Based on the similarity between the brain and the neural retina, the aim of the current study was to evaluate the expression of CYP46A1 in the mammalian retina. RT-PCR analysis of CYP46A1 in bovine samples revealed the highest expression in the neural retina. The retinal pigment epithelium expressed CYP46A1 gene at a low level while the ciliary body showed no expression. Immunohistochemical evaluation of the posterior pole of rat retina showed that the protein is specifically expressed in neurons, whereas cone-rods photoreceptors were negative for CYP46A1 staining. The metabolite produced by CYP46A1, 24S-hydroxycholesterol, was almost exclusively found in neural retina, the concentration therein being more than 10-fold higher than in the retinal pigment epithelium or the ciliary body. The results of the current study are consistent with our primary hypothesis: the neural retina specifically expresses cholesterol-24S-hydroxylase, a metabolizing enzyme responsible for the removal of cholesterol in neurons. Based on the link between cholesterol-24S-hydroxylase, 24S-hydroxycholesterol, and neurologic disorders, CYP46A1 may be a valuable gene candidate for retinal pathologies like age-related macular degeneration or glaucomas, and 24S-hydroxycholesterol may be involved in the onset of the degenerative processes in these diseases.

1. We developed a novel method to isolate nonpigmented epithelial (NPE) cells from porcine eyes in order to examine Na,K-ATPase responses to nitric oxide (NO) donors specifically in the epithelium. 2. Cells were treated with NO donors and other test compounds for 20 min prior to Na,K-ATPase activity measurement. 3. NO donors, sodium nitroprusside (SNP, 1 microM-1 mM), sodium azide (100 nM-1 microM) and S-nitroso-N-acetylpenicillamine (1 microM-1 mM) caused significant concentration-dependent inhibition of Na,K-ATPase activity. Detection of nitrite in the medium of L-arginine and SNP-treated NPE confirmed NO generation. 4. Concentration-dependent inhibition of Na,K-ATPase was also obtained by L-arginine (1-3 mM), a physiological precursor of NO and 8p-CPT-cGMP (1-100 microM), a cell permeable analog of cGMP. The L-arginine effect was abolished when the NO synthesizing enzyme, NO-synthase, was inhibited by L-NAME (100 microM). 5. The inhibitory effect of SNP or sodium azide on Na,K-ATPase activity was suppressed by soluble guanylate cyclase (sGC) inhibitors, ODQ (10 microM) or methylene blue (10 microM). 6. The inhibitory effect of 8p-CPT-cGMP on Na,K-ATPase was abolished by protein kinase G (PKG) inhibitors, H-8 (1 microM) and H-9 (20 microM), but not by the protein kinase A (PKA) inhibitor H-89 (100 nM). H-8 and H-9 partially suppressed the inhibitory effect of SNP on Na,K-ATPase. 7. Taken together the results indicate that Na,K-ATPase inhibition response to NO donors involves activation of sGC, generation of cGMP and activation of PKG. These findings suggest that Na,K-ATPase inhibition in NPE may contribute to the ability of NO donors to reduce aqueous humor secretion.

Purpose: To determine the effect of latanoprost on the expression of human matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in the ciliary body., Methods: Total RNA was isolated, and qualitative RT-PCR was performed to detect the mRNA of MMPs and TIMPs in human ciliary body tissue and explant cultures of ciliary body smooth muscle (CBSM) cells. CBSM cell cultures were treated with vehicle control or latanoprost acid for 24 hours. Quantitative RT-PCR of cell cultures from five different donors was performed to determine relative changes in expression. GAPDH served as an endogenous control., Results: The mRNA of MMP-1, -2, -3, -11, -12, -14, -15, -16, -17, -19, and -24 as well as TIMP-1 to -4 were found in ciliary body tissue and CBSM cells. MMP-9 was present after latanoprost treatment. In control CBSM cells, the relative expression of MMP mRNA was MMP-2 and -14 > MMP-24 > MMP-1, -11, -15, -16, and -19 > MMP-3 and 17, > MMP-12. The relative expression of TIMP mRNA was TIMP-2 > TIMP-1 > TIMP-3 > TIMP-4. Latanoprost increased MMP-3 (in three of five cultures), MMP-17 (in four of five cultures), and TIMP-3 (in all five cultures); MMP-1, -2, -12, -14, -15, and -16 and TIMP-4 were downregulated., Conclusions: The transcription of the genes for MMP-3 and -17 is increased by latanoprost treatment. MMP-9 is present after latanoprost treatment and may also mediate ECM changes. TIMP-3 is upregulated and may compensate for the increase in MMPs. These coordinated changes could be expected to mediate the latanoprost-induced alteration of ECM in the ciliary body.

Purpose: To verify the capability of rabbit and rat ciliary body to synthesize and secrete ceruloplasmin., Methods: Isolated ciliary body (CB) was cultured in the presence of [35S]-methionine, and the incubation medium was processed for immunoprecipitation. Total RNA from CB was processed for RT-PCR, and the amplification products were sequenced. Also, sections of CB were immunostained for the localization of ceruloplasmin., Results: A labeled peptide, having a molecular weight of about 135 kDa, the expected size of ceruloplasmin, was immunopurified in the incubation media from both animal species. The RT-PCR and sequencing experiments detected the presence of ceruloplasmin mRNA in rat samples. Both layers of rabbit and rat ciliary epithelium (CE) exhibited ceruloplasmin reactivity after immunohistochemical processing., Conclusions: Taken altogether, these results indicate the CB, particularly its epithelium, as one of the possible sources of the ocular intrinsic ceruloplasmin.

The uveitogenic bacterial cell wall component muramyl dipeptide (MDP) is apoptogenic in rabbit kidney cells. The purpose of this investigation was to assess the cytotoxic activity of MDP and staurosporine (STSP; induces cultured corneal and lens cells apoptosis) in rabbit ciliary body tissue. Anterior uveitis was determined by clinical symptoms and increased aqueous humor (AH) protein. Ciliary body tissue was assessed for histological changes, caspase-3 activity, dye uptake, distribution of immunoreactive caspase-3 and DNA ladders at 4 and 6 hours postinjection. Increases in caspase-3 activity, APOPercentage dye uptake, and localization of immunoreactive caspase-3 in ciliary epithelial cells were associated with ciliary cysts of detached nonpigmented epithelial (NPE) cells, as well as apoptotic and necrotic DNA ladders in ciliary body tissues from eyes injected with MDP and/or STSP. The results suggest that intracameral injection of the bacterial components MDP and STSP can induce acute endophthalmic changes in uveal tissue including formation of ciliary body, NPE and pigmented epithelial (PE) cell apoptosis, and ciliary body tissue necrosis.

This study investigated the heme oxygenase-1 (HO-1) and the endotoxin-induced uveitis (EIU) in diabetic streptozotocin (STZ)-hyperglycemic rats. STZ-hyperglycemic rats had impaired levels of the enzyme HO-1 within the ciliary bodies if compared with the nondiabetic rats. STZ-hyperglycemic rats also predisposed the eye to produce high levels of both the cytokines IL-1beta and CXCL8. Subsequent EIU further and significantly (P < .01) increased the cytokines production, an effect partly prevented by hemin treatment. Most importantly, hemin, an inducer of heme oxygenase expression and activity, recovered the huge number of infiltrated polymorphonuclear leukocytes PMN within the ciliary bodies associated with STZ-hyperglycemic state and EIU damage. Impairment of the stress-sensitive enzyme HO-1 in STZ-hyperglycemic rats increases and prolongs the inflammatory response to EIU.

Purpose: To compare the reactive proliferation of non-pigmented ciliary epithelial cells in patients with cyclitis, or after cyclophotocoagulation and cyclocryocoagulation, and the cultured non-pigmented ciliary epithelial cells from adult pigs., Methods: Porcine ciliary epithelial cells were cultured and non-pigmented ciliary epithelial cells were isolated. Detection of DNA synthesis, morphological observation by a phase contrast microscope and a transmission electron microscope, and staining of senescence-associated beta-galactosidase were carried out., Results: The cells proliferated without showing contact inhibition of growth or reconstitution of epithelial morphology. With a decrease of proliferative activity, the cultured cells expressed senescence-associated beta-galactosidase. Although DNA synthesis persisted for a long time, some cells in later culture periods showed morphologically abnormal nuclei or plural nuclei indicating dysfunction of cell division, or apoptotic features., Conclusion: The uncontrolled growth and loss of the epithelial nature of non-pigmented ciliary epithelial cells in vitro resembles the process of formation of cyclitic membrane and proliferation of ciliary epithelium after cyclophotocoagulation and cyclocryocoagulation in patients. Observatin of the behavior of cultured non-pigment epithelial cells could aid in understanding the mechanism of cyclitic membrane formation.

Purpose: Studies were designed to evaluate the cellular mechanisms associated with prostaglandin (PG)F(2alpha)-induced matrix metalloproteinase (MMP)-2 secretion from human ciliary muscle (HCM) cells., Methods: The secretion and activity of MMP-2 was determined by Western blot analysis and zymography, using conditioned medium and HCM cells. ERK1/2 activity was measured by in-gel kinase assay and Western blot analysis with anti-phospho-ERK1/2 antibodies., Results: PGF(2alpha) increased the secretion of MMP-2 in a dose-dependent manner with an EC(50) of 2.7 x 10(-8) M. The addition of 1 muM PGF(2alpha) also increased MMP-2 secretion in a time-dependent manner with maximum secretion occurring at 4 hours after administration. At 4 hours, the maximum increase in MMP-2 secretion and activity were 112% +/- 32% and 88% +/- 18%, respectively. The secretory action of PGF(2alpha) was inhibited by pretreatment with a protein kinase C (PKC) inhibitor, chelerythrine chloride; the FP receptor antagonist, AL-8810; and the MEK inhibitor, PD-98059. The addition of PGF(2alpha) and latanoprost acid increased ERK1/2 activity by 117% +/- 12% and 75% +/- 9%, respectively. The PGF(2alpha)- and latanoprost-acid-induced ERK1/2 activation was blocked by the presence of PKC inhibitors and downregulation of PKC by prolonged incubation with a phorbol ester., Conclusions: These data provide evidence that FP receptor activation leads to an increase in the secretion and activation of MMP-2 through PKC- and ERK1/2-dependent pathways. FP-agonist-induced activation of ERK1/2 was blocked by PKC inhibitors, indicating that PKC activation is required for ERK1/2 activation and MMP-2 secretion from HCM cells. In the ciliary muscle, the functional responses to ERK1/2 activation include secretion of MMP-2, supporting the hypothesis that increases in uveoscleral outflow facility induced by PG administration involves the secretion and activation of MMP-2.

Müller cells, the radially oriented dominant macroglial cells of the retina, are known to contain abundant glycogen as well as the key enzyme for its degradation, glycogen phosphorylase (GP), but the expressed isozyme pattern is unknown. To elucidate the isoform expression pattern, specific antisera directed against the brain (BB) and muscle (MM) isoforms of GP were applied to retinal sections, isolated Müller cells, and sections of the optic nerve. We show that Müller cells of rat, rabbit, guinea pig, and mouse retina exclusively express the BB isoform. Astrocytes of rat and rabbit optic nerve, as well as retina express only the BB isoform. In contrast, astrocytes in the brain and spinal cord as well as the epithelial cells of the pars caeca and of the ciliary body express both the BB and MM isoform. This result may indicate some differences in the role of glycogen in retinal macroglia and brain astrocytes, reflecting a local specialization of macroglia in the retina proper., (copyright (c) 2004 Wiley-Liss, Inc.)

Purpose: Two isoforms of cyclooxygenase (COX-1 and COX-2) exist. To determine in vivo effects of the intravenous administration of FR122047 (a selective COX-1 inhibitor), FR188582 (a selective COX-2 inhibitor), diclofenac sodium or dexamethasone phosphate disodium on prostaglandin-E2 (PGE2)-induced aqueous flare elevation and mRNA levels for COX-1 and COX-2 in pigmented rabbits., Methods: To produce aqueous flare elevation in rabbits, PGE2, 25 microg/ml, was applied to the cornea with the use of a glass cylinder. FR122047, FR188582, diclofenac sodium or dexamethasone phosphate disodium was intravenously injected before PGE2 application. Aqueous flare was measured with a laser flare-cell meter. The mRNA levels for COX-1 and COX-2 in the iris-ciliary body were determined by real-time polymerase chain reaction., Results: FR122047, FR188582 and diclofenac sodium (15 micromol/kg each) injected intravenously 30 min before PGE2 application inhibited 29 +/- 5, 40 +/- 12 and 50 +/- 9% of aqueous flare elevation, respectively. Simultaneous injection of FR122047 (15 micromol/kg) and FR188582 (15 micromol/kg) 30 min before PGE2 application inhibited 61 +/- 8% of flare elevation. Dexamethasone phosphate disodium (15 micromol/kg) injected intravenously 300 min before PGE2 application inhibited 68 +/- 8% of aqueous flare elevation. Less than 3-fold changes in mRNA levels for COX-1 and COX-2 in the iris-ciliary body were noted after PGE2, FR122047, FR188582, diclofenac sodium or dexamethasone phosphate disodium treatment., Conclusion: It is possible that enzyme activities of both COX-1 and COX-2 may be involved in the mechanism of PGE2-induced aqueous flare elevation in pigmented rabbits., (Copyright 2004 S. Karger AG, Basel.)

The retinal pigment epithelium (RPE) and the choriocapillaris are affected early in the retinopathy associated with long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency. RPE in culture possesses the machinery needed for mitochondrial fatty acid beta-oxidation in vitro. To further elucidate pathogenesis of LCHAD retinopathy, we performed immunohistochemistry of the human eye and brain with antibodies to beta-oxidation enzymes. Human eye and brain sections were stained with antibodies to medium-chain (MCAD) and very long-chain acyl-CoA dehydrogenase (VLCAD), short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), and mitochondrial trifunctional protein (MTP) harboring LCHAD. Antibodies to 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) and cytochrome c oxidase subunit I (COX I) were used as a reference. VLCAD, MTP, MCAD, SCHAD, MHBD, and COX I antibodies labeled most retinal layers and tissues of the human eye actively involved in oxidative metabolism (extraocular and intraocular muscle, the RPE, the corneal endothelium, and the ciliary epithelium). MTP and COX I antibodies labeled the inner segments of photoreceptors. The choriocapillaris was labeled only with SCHAD and MCAD antibodies. In the brain, the choroid plexus and nuclei of the brain stem were most intensely labeled with beta-oxidation antibodies, whereas COX I antibodies strongly labeled neurons in several regions of the brain. Mitochondrial fatty acid beta-oxidation likely plays a role in ocular energy production in vivo. The RPE rather than the choriocapillaris could be the critical affected cell layer in LCHAD retinopathy. Reduced energy generation in the choroid plexus may contribute to the cerebral edema observed in patients with beta-oxidation defects.

Purpose: To clarify the mechanism by which bunazosin hydrochloride (BZ), a selective alpha1-adrenoceptor antagonist, increases uveoscleral outflow., Methods: The effects of BZ on matrix metalloproteinase (MMP) activities in cultured monkey ciliary muscle cells, and on phenylephrine hydrochloride-induced constriction in bovine ciliary muscles, were examined. Also, the possible additive ocular hypotensive effects of BZ and latanoprost (LP) were evaluated in ocular normotensive monkeys., Results: Although BZ at 10 to 10 M did not increase MMP-2, -3, and -9 activities in the culture medium of ciliary muscle cells, BZ at 10 to 10 M inhibited phenylephrine hydrochloride-induced constriction in ciliary muscles. The maximal reduction in intraocular pressure of concomitant administration of BZ and LP was greater than that of BZ alone and tended to be greater than that of LP alone., Conclusion: These findings, in normotensive monkeys, indicate that the mechanism whereby BZ increases uveoscleral outflow is independent of an effect on MMPs and is partly due to relaxation of the ciliary muscle. This effect is different from that of LP, which might help to explain the finding that topical concomitant administration of BZ and LP increased AUC(0-6h) value (IOP reduction) to 201% and 145% of BZ and LP given alone, respectively.

This study was undertaken to investigate the effect of some cannabinoid agonists on the bovine ciliary muscle. Both anandamide and CP 55,940 (cis-3-(2-hydroxy-4-(1,1-dimethyl heptyl) phenyl)-trans-4-(3-hydroxypropyl) cyclohexanol) produced a concentration-dependent contractile response in ciliary muscle. These responses were inhibited by SR 141716A (N-[piperidin-1-yl]-5-(4-cholophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) (0.1 and 1 microM) but not by SR 144528 (N-[1S)-endo-1,3,3-trimethyl bicyclo[2.2.1] heptan-2-yl] 5-(4-chloro-3-methylphenyl)-1-(4 methoxy benzyl)-pyrazole-3-carboxamide) (1 and 10 microM). A preincubation with G(i/o) protein inhibitor pertussis toxin (500 ng/ml) for 20 min inhibited the contractile action of anandamide and CP 55,940. In addition, the phospholipase C inhibitor U73122 (1[6-[[(17 beta)-3-methoxyestra-1,3,5(10)-trien-17-yl] amino] hexyl]-1H-pyrrole-2,5-dione) blocked the anandamide- and CP 55,940-induced contractions, whereas the protein kinase C activator, phorbol 12,13 dibutyrate (PDBu) significantly potentiated the contractions evoked by cannabinoid receptor agonists. We evaluated the binding of [(3)H]CP 55,940, which specifically labelled a single class of cannabinoid sites with affinity in low subnanomolar range (K(d)=0.6 nM) and the maximal number of binding sites of 1243 fmol/mg protein. Binding of [(3)H]CP 55,940 was inhibited by ligands having a major selectivity for cannabinoid (CB(1)) receptors. These findings provide strong evidence of the involvement of cannabinoid CB(1) receptors promoting contraction in the bovine ciliary muscle. Furthermore, the action of cannabinoid receptor agonists appears to be mediated via phospholipase C. These data also contribute to elucidate the cannabinoid CB(1) receptor pivotal role in the modulation of intraocular pressure and to show that cannabinoid receptor agonists may be regarded as potential antiglaucoma agents.

We have recently discovered a splice variant of choline acetyltransferase (ChAT) mRNA and designated the variant protein pChAT because of its preferential expression in peripheral neuronal structures. In this study, the presence of pChAT in rat iris was examined by immunohistochemistry and Western blot using a pChAT antiserum, in combination with RT-PCR analysis and ChAT enzyme assay. For comparison, the conventional ChAT (cChAT) was studied in parallel. By pChAT immunohistochemistry, intense labeling was found to occur in nerve fibers of the iris and in neurons of the ciliary and trigeminal ganglia. Denervation studies, analyzed by semiquantitative morphometry, indicated that these iridial pChAT fibers originated about half from the ciliary ganglion and the other half from the trigeminal ganglion. The presence of pChAT protein in the iris and trigeminal ganglion was confirmed by Western blot. The expression of pChAT mRNA in the ciliary and trigeminal ganglia was proved by RT-PCR. Although cChAT protein and mRNA were detected in the ciliary ganglion, neither was detectable in the trigeminal ganglion. The contributions of the ciliary and trigeminal ganglia to the iridial ChAT enzyme activity were verified by the present ChAT assay. Here, we provide evidence that iridial pChAT nerves are composed of postganglionic parasympathetic efferents from the ciliary ganglion and, more interestingly, somatic sensory afferents of the trigeminal ophthalmic nerve., (Copyright 2004 Wiley-Liss, Inc.)

Endotoxin-induced uveitis (EIU) is an animal model of acute ocular inflammation. Cytokines, chemokines, and nitric oxide (NO) have been reported to play important roles. We have determined whether heme oxygenase (HO)-1, a heat shock protein, can suppress EIU. EIU was induced by a footpad injection of lipopolysaccharide (LPS) in male Lewis rats. Hemin, an inducer of HO-1, was injected intraperitoneally 1 hr prior to the LPS injection. HO-1 and HO-2 expression in the iris-ciliary body (ICB) was studied by real time PCR and Western blot analysis. The number of infiltrating cells and the protein concentration in the aqueous humor (AqH) were evaluated by microscopy and by protein assay. The expression of inducible nitric oxide synthase (iNOS), interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and IL-1beta mRNA was determined by real time PCR. The concentration of nitrate plus nitrite, and levels of IL-6 and TNF-alpha in the AqH were also evaluated by Griess reagents and by enzyme-linked immunosorbent assay, respectively. The expression of HO-1 mRNA and protein, induced by LPS, was enhanced significantly by pre-injection of hemin (P<0.001). HO-2 was constitutively present in the ICB and was not up-regulated by LPS or by hemin. The number of infiltrating cells and the concentration of protein in the AqH was significantly elevated by LPS injection, and hemin significantly reduced the number of cells and the protein concentration (P<0.0001). The expression of iNOS and IL-6 mRNA and protein were down-regulated by hemin (P<0.001). Hemin is effective in inducing HO-1 and in reducing the ocular inflammation induced by LPS probably by down-regulating NO and pro-inflammatory cytokine expression.

Phospholipase C induced phosphoinositide (PI) turnover, intracellular Ca(2+) ([Ca(2+)](i)) mobilization and mitogen-activated protein (MAP) kinase activation by FP-class prostaglandin analogs was studied in normal human ciliary muscle (h-CM) cells. Agonist potencies obtained in the PI turnover assays were: travoprost acid ((+)-fluprostenol; EC(50) = 2.6 +/- 0.8 nM) > bimatoprost acid (EC(50) = 3.6 +/- 1.2 nM) > (+/-)-fluprostenol (EC(50) = 4.3 +/- 1.3 nM) >> prostaglandin F(2 alpha) (PGF(2 alpha)) (EC(50) = 134 +/- 17 nM) > latanoprost acid (EC(50) = 198 +/- 83 nM) > S-1033 (EC(50) = 2930 +/- 1420 nM) > unoprostone (EC(50) = 5590 +/- 1490 nM) > bimatoprost (EC(50) = 9600 +/- 1100 nM). Agonist potencies in h-CM cells correlated well with those previously obtained for the cloned human ciliary body-derived FP receptor (r = 0.96, p< 0.001) and that present on h-TM cells (r = 0.94, p< 0.0001). Travoprost acid, PGF(2 alpha) and unoprostone also stimulated [Ca(2+)](i) mobilization in h-CM cells with travoprost acid being the most potent agonist. MAP kinase activity was stimulated in the h-CM cells with the following rank order of activity (at 100 nM): travoprost acid > PGF(2 alpha) > latanoprost acid > PGD(2) > bimatoprost > latanoprost = bimatoprost acid = fluprostenol > PGE(2) = S-1033 > unoprostone > PGI(2). The PI turnover, [Ca(2+)](i) mobilization and MAP kinase activation induced by several of these agonists was blocked by the FP receptor antagonist, AL-8810 (11 beta-fluoro-15-epiindanyl PGF(2 alpha)) (e.g. K(i) = 5.7 microM versus PI turnover). These studies have characterized the biochemical and pharmacological properties of the native FP prostaglandin receptor present on h-CM cells using three signal transduction mechanism assays and a broad panel of FP-class agonist analogs (including free acids of bimatoprost, travoprost and latanoprost) and the FP receptor antagonist, AL-8810.

Endothelin-1 (ET-1) (1-100 nM) decreases the activity of Na,K-ATPase, a key enzyme responsible for aqueous humor formation, in transformed human non-pigmented ciliary epithelial (HNPE) cells. The present study sought to determine if ET-1 alters the expression of the catalytically active alpha subunit of Na,K-ATPase in HNPE cells and identify mechanisms underlying these effects. We report that acute (15 and 30 min) treatment with ET-1 results in an increase in mRNA expression of the alpha 1 subunit of Na,K-ATPase. Similar to ET-1's effects, ouabain (100 microM), a selective inhibitor of Na,K-ATPase, and monensin (10 microM), a sodium ionophore, also increased Na,K-ATPase expression in HNPE cells. The increase in Na,K-ATPase expression by short-term treatment with ouabain and monensin was dependent on their ability to elevate intracellular sodium concentrations. However, acute ET-1 treatment mediated increase in Na,K-ATPase expression was independent of changes in intracellular sodium. A prolonged (24 hr) ET-1 treatment results in an increase in both mRNA and protein levels of the alpha 1 subunit of Na,K-ATPase. These observations suggest that ET-1 could play a homeostatic role in modulating aqueous humor formation by having differential effects on the activity and expression of Na,K-ATPase by the ciliary epithelium in the eye.

Purpose: To investigate the expression of both the message and function of ENPP1 (a member of the ectonucleotide pyrophosphatase/phosphodiesterase family, also known as PC-1), NTPD1 (a member of the ectonucleoside 5'-triphosphate diphosphohydrolase family, CD39), and ecto-5'-nucleotidase (CD73) in rabbit ciliary body nonpigmented epithelial (NPE) cells., Methods: Reverse transcription-polymerase chain reaction (RT-PCR) analysis was used to reveal the presence of mRNAs of ectonucleotidases in NPE cells. Real-time fluorescence ratio imaging of the intact fura-2-loaded NPE cells was used to record changes in the intracellular calcium concentration., Results: RT-PCR analysis revealed the expression of mRNAs for ENPP1, NTPD1, and ecto-5'-nucleotidase, but not NTPD2 (ecto-ATPase, or CD39L1), in the rabbit NPE cells. The ENPP1 inhibitor pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), and to a lesser degree the nonspecific ectonucleotidase antagonist 6-N,N-diethyl-beta-gamma-dibromomethylene-D-adenosine 5-triphosphate (ARL 67156), reduced the [Ca(2+)](i) increase elicited by the combination of acetylcholine (ACh) and cAMP. However, both inhibitors significantly enhanced the [Ca(2+)](i) increase generated by uridine triphosphate (UTP). The ecto-5'-nucleotidase inhibitor alphabeta-meADP significantly diminished the [Ca(2+)](i) increase evoked by ACh+cAMP, but not that generated by UTP. The A(1)-specific adenosinergic receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) significantly blocked the response to ACh+cAMP., Conclusions: These observations suggest that rabbit NPE cells possess at least three distinct ectonucleotidases capable of catalyzing the stepwise hydrolysis of adenine and pyrimidine nucleotides, as well as cAMP, thus shaping the purinergic-receptor-coupled signaling in these cells.

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.

The CYP39A1 oxysterol 7alpha-hydroxylase preferentially catalyzes the 7alpha-hydroxylation of 24-hydroxycholesterol and has been suggested to play a role in the alternative bile acid synthesis pathway in the liver. The presence of CYP39A1 oxysterol 7alpha-hydroxylase has been reported only in the liver. To investigate the physiologic characteristics of the ciliary processes in bovine ocular tissues, we raised a mAb, 42C, against nonpigmented epithelial (NPE) cells, which have tight junctions that act as a blood-aqueous barrier and are involved in producing aqueous humor and maintaining ocular homeostasis. Immunohistochemical analysis showed that 42C antibody reacted intensely with an antigen in the NPE cells of the ciliary processes but not with other ocular tissues. The SDS-PAGE profile of immunoaffinity-purified antigens from bovine ciliary processes showed a predominant protein of molecular mass of 44.0 kDa. The amino acid sequence of this antigenic protein was identical to human CYP39A1 oxysterol 7alpha-hydroxylase. Immunoreactivity with 42C antibody was found only in hepatocytes and ocular tissues. These data suggest a new physiologic function for the CYP39A1 oxysterol 7alpha-hydroxylase in addition to the production of bile acids and provide new insight into the physiologic role of the ciliary NPE cells concerning the metabolism of sterols in the eye.

Purpose: To investigate the localization and the activity of membrane dipeptidase (MDP) in the bovine eye., Methods: A monoclonal antibody (mAb), 49C mAb, raised against bovine ciliary process was used to examine the localization of MDP. Conversion of leukotriene (LT)D4 to LTE4 was evaluated by enzyme-linked immunosorbent assay for LTE4. Hydrolytic activity (beta-lactamase activity) was evaluated with a fluorometric assay. To clarify the contribution of MDP to conversion of LTD4 and beta-lactamase activity, we separated MDP from other enzymes by 49C mAb-conjugated gel., Results: The antigenic molecule of 49C mAb was shown to be MDP by amino acid sequencing. MDP was immunohistochemically detected in the ciliary pigmented and nonpigmented epithelial cells. Conversion of LTD4 to LTE4 in the ciliary process was much greater than that of the neural retina (NR). beta-Lactamase activity in the ciliary process was apparent, but that in the NR or the retinal pigment epithelium was negligible. Approximately 100% of beta-lactamase activity in the ciliary process was catalyzed by the 49C mAb-bound fraction. Conversion of LTD4 was catalyzed by the 49C mAb-bound fraction (55% of total activity) and by the unbound fraction (45% of total activity)., Conclusions: This study produced the first evidence of the presence of MDP in ciliary epithelial cells. The ciliary epithelium converts LTD4 to LTE4 and shows beta-lactamase activity. Conversion of LTD4 is catalyzed by at least two enzymes, and a major part of the conversion is induced by MDP.

Purpose: To determine the effect of treatment with latanoprost on the tissue inhibitors of metalloproteinase (TIMP)-1 and -2 in cultured human ciliary muscle (HCM) cells., Methods: Confluent serum-starved HCM cells were exposed to increasing concentrations of latanoprost acid (LA, 1 nM to 10 micro M) for 6, 18, and 24 hours. TIMP-1 and -2 mRNA transcripts were evaluated by RT-PCR. Gelatin zymography was used to measure changes in the amount of matrix metalloproteinase (MMP) in the culture medium. To evaluate the potential role of PKC, HCM cells were treated with phorbol 12-myrisate 13-acetate (PMA) in the absence or presence of the PKC inhibitor bisindolylmaleimide I (Bis I) or the PKA inhibitor KT5720. Data were quantitated by densitometry and statistically analyzed with the Student-Newman-Keuls test., Results: TIMP-1 and -2 mRNA transcripts and proteins were detected in primary cultures of HCM cells. TIMP-1 mRNA levels were unchanged at 6 hours, but increased 45% +/- 17% and 54% +/- 13% in cultures exposed for 18 hours to 1 and 10 micro M LA, respectively (n = 3). In contrast, 6 hours of exposure to LA increased expression of TIMP-2 mRNA by up to 11.3% +/- 0.2% (n = 3). However, no significant induction of TIMP-2 mRNA was observed at either 18 or 24 hours (n = 3). TIMP-1 protein was significantly increased in cultures exposed to LA for 18 and 24 hours. In contrast, TIMP-2 protein expression was insignificantly different from control cultures at 6, 18, and 24 hours of treatment. HCM cells exposed to PMA for 24 hours produced similar increases in TIMP-1 mRNA levels, as seen with latanoprost (n = 5). However, no significant induction of TIMP-2 mRNA was observed. Zymographic analysis of the media from these cultures confirmed dose-dependent increases of MMP-1 at 6, 18, and 24 hours, whereas dose-dependent increases in MMP-2 were seen only after 24 hours' exposure to LA (n = 3). TIMP-1 protein levels were increased 27% +/- 9.3% and 15% +/- 11% in the media of cells exposed for 24 hours to 100 nM LA and 100 nM PMA, respectively (n = 5). The increases in TIMP-1 protein induced by LA were essentially eliminated by Bis I (n = 3) and unaffected by KT5720 (n = 3)., Conclusions: For the most part, TIMP-1, and not TIMP-2, contributes to regulation of MMP within the uveoscleral outflow pathway after exposure to latanoprost. Moreover, this induction appears to be meditated by activation of PKC.

Prohormone convertases PC1 and PC2 are enzymes involved in the intracellular processing of pro-neurotensin/neuromedin N (pro-NT/NN) through the regulated secretory pathway. In this study, we present evidence of the differential gene expression of pro-NT/NN, pro-PC1 and pro-PC2 in two cell lines established from the neuroendocrine ocular ciliary epithelium. Dexamethasone and forskolin were found to synergistically up-regulate NT/NN mRNA expression in both cell types. The pigmented cells released NT, and this release was enhanced by agents that induced its biosynthesis. In contrast, nonpigmented cells exhibited a significantly reduced neurotensin secretion in response to inducers, leading to an accumulation of the peptide. PC1 and PC2 mRNA expression was induced in a cell-specific manner by the same agents that enhanced pro-NT/NN biosynthesis. These results demonstrate cell-specific processing of pro-NT/NN by the ciliary epithelium., (Copyright 2002 Elsevier Science Ireland Ltd.)

Background: To determine whether nitric oxide synthase (NOS) protein expression can be modulated by forskolin in porcine ciliary processes., Method: Western blot analysis using mouse monoclonal antibodies against NOS I (neuronal NOS or nNOS), NOS II (macrophage NOS or macNOS), and NOS III (endothelial NOS or eNOS) was performed in porcine ciliary processes. Expression of NOS I and II was also assessed in the presence or in the absence of 1 microM forskolin exposure (4 and 24 hours)., Results: All three NOS isoforms could be detected in isolated porcine ciliary processes. Protein expression for NOS I was about 6 times higher than for NOS III and 15 times higher than for NOS II. In comparison to controls, after forskolin exposure, NOS I protein expression was increased by about 1.5-fold. No change could be observed in NOS II protein expression after forskolin exposure., Conclusions: Nitric oxide is involved in transepithelial fluid transport of different organs (lung, kidney, colon, etc.) and is suspected to play a role in ciliary processes by modulating the aqueous humor production. The present study indicates that forskolin (which increases cAMP production) modulates NOS I protein expression in isolated porcine ciliary processes. These results suggest that selective inhibition of NOS I could be tried for reducing aqueous humor production. The clinical relevance requires further investigations.

Purpose: The present study was undertaken to determine whether treatment of ciliary muscle cells with the prostaglandin (PG) analogue latanoprost acid alters transcription of mRNA for matrix metalloproteinase (MMP)-1, -2, -3, and -9., Methods: Human ciliary smooth muscle cell cultures were grown to confluence and treated for 24 hours with medium supplemented with latanoprost acid or vehicle. Total RNA was then isolated, and the expression of mRNAs for MMP-1, -2, -3, and -9 were determined using Taqman and energy-transfer real-time PCR analyses. All results were normalized according to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in each sample., Results: Specificity and linearity of each real-time PCR assay were confirmed by electrophoresis and serial dilution analysis of oligonucleotides containing the amplicon sequence. Addition of latanoprost acid for 24 hours increased expression of MMP-1 by 3- to 13-fold in three of five primary ciliary muscle lines. Addition of 8, 40, and 200 nM latanoprost acid for 24 hours increased MMP-1 mRNA in a dose-dependent manner. Analysis of cultures exposed to 200 nM latanoprost acid for 4, 6, 12, or 24 hours revealed an increase and then a decline of MMP-1 mRNA, with peak expression at 6 to 12 hours after initiation of treatment. Parallel assessments of RNA from ciliary muscle cultures exposed to latanoprost acid for 24 hours revealed increased MMP-1, -3, and -9 mRNAs and reduced MMP-2 mRNA, when compared with RNA from vehicle-treated cultures., Conclusions: Latanoprost acid induced a dose-dependent increase of MMP-1, -3, and -9 gene transcription in cultured human ciliary smooth muscle cells. These results are consistent with increased MMPs contributing to the increased uveoscleral outflow facility observed after topical latanoprost.

Purpose: The important role of ET-1 in vasoconstriction has been shown for the vasoregulation in the retina, choroid and optic nerve. ET-1 induced vaso-constriction, however, can be treated effectively at the level of endothelin-1 converting enzyme (ECE-1) by ECE-1 inhibitors because ECE-1 converts biologically almost inactive big endothelin to endothelin-1 (ET-1), the most potent vasoconstrictor known. The purpose of this study was to clarify the anatomical distribution of ECE-1 in the human eye., Methods: 11 post-mortem eyes were fixed in 4% formalin and embedded in paraffin. 4 microm thin sections were analyzed immuno-histochemically using a self-produced monoclonal primary antibody against human ECE-1 and a polyclonal alpha-actin antibody for comparison., Results: ECE-1 -IR was demonstrated in the corneal epithelium, vascular smooth muscle and endothelial cells, and in the non-vascular smooth muscle cells of the ciliary body, the dilator and sphincter muscle of the iris., Conclusions: A strong immunoreactivity for ECE-1 can be found in the blood vessels of the retina, optic nerve and choroid. Therefore, it should be possible to treat ET-1-induced vaso-constriction in the eye using ECE-1 inhibitors, especially in diseases like hypertensive vasculopathy, vasospasm, vaso-occlusions or low tension glaucoma.

Purpose: To characterize in vivo changes of adenylate cyclase activity in rabbit ciliary processes during the circadian cycle., Methods: After removal of vitreous, lens, retina and choroid from freshly enucleated rabbit eyes, the anterior segment and attached sclera was chilled in cold buffer containing the non-selective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Then ciliary processes were excised and homogenized in ice cold trichloro-acetic acid (TCA) 2.5 min after IBMX treatment. Increased cyclic AMP in response to PDE inhibition was measured in ciliary processes at six times during the circadian cycle, after preganglionic section of the cervical sympathetic trunk (decentralization, DX), and after topical instillation of hydroxyamphetamine, timolol, brimonidine, rauwolscine or a soluble derivative of forskolin., Results: The increase of rabbit ciliary process cyclic AMP levels in response to PDE inhibition with IBMX was enhanced at night. Much of the enhanced nocturnal increase persisted in constant dark and was blunted by DX. Topical instillation of hydroxyamphetamine enhanced the increase during the day; whereas, timolol, a beta-adrenergic antagonist, or brimonidine, an alpha2-adrenergic agonist, blunted the IBMX induced increase. Neither instillation of rauwolscine, an alpha2-adrenergic antagonist, nor the soluble forskolin derivative enhanced the increase of cyclic AMP after IBMX., Conclusions: The technique reported here can be used to estimate ciliary process adenylate cyclase activity in vivo. There is a circadian rhythm of adenylate cyclase activity in rabbit ciliary processes that is driven in part by ocular sympathetic input and stimulation of beta-adrenergic receptors.

Purpose: Primary open-angle glaucoma (POAG) is the predominant form of chronic glaucoma, but the underlying pathologic mechanisms are largely unknown. Because prostaglandins (PGs) have been introduced into POAG treatment with remarkable success, this study was undertaken to investigate whether a change in the expression of the PG-synthesizing enzymes cyclooxygenase (COX)-1 and -2 might be involved in the pathogenesis of POAG., Methods: Expression of COX-1 and -2 was assessed by confocal laser microscopy, immunohistochemistry, Western blot analysis, and real-time RT-PCR in human eyes with different forms of glaucoma (primary open-angle, angle-closure, congenital juvenile, and steroid-induced), as well as in age-matched control eyes. Additionally, PGE2 was measured in aqueous humor by means of an enzyme-linked immunoassay as a product of COX activity., Results: In normal eyes, ocular COX-1 and -2 expression were largely confined to the nonpigmented secretory epithelium of the ciliary body. By immunohistochemistry and real-time RT-PCR, COX-2 expression was completely lost in the nonpigmented secretory epithelium of the ciliary body of eyes with end-stage POAG, whereas COX-1 expression was unchanged. By immunohistochemistry, in the ciliary bodies of eyes in five patients with diagnosis of early POAG, eyes in two had complete loss of COX-2 expression and in three showed only a few remaining scattered COX-2-expressing cells. COX-2 expression in the ciliary body was also lost in patients with steroid-induced glaucoma and was reduced in patients receiving topical steroid treatment. Eyes of patients with either congenital juvenile or angle-closure glaucoma showed COX-2 expression indistinguishable from control eyes. Aqueous humor of eyes with POAG contained significantly less PGE2 than control eyes., Conclusions: Both cyclooxygenase isoforms are constitutively expressed in the normal human eye. Specific loss of COX-2 expression in the nonpigmented secretory epithelium of the ciliary body appears to be linked to the occurrence of POAG and steroid-induced glaucoma.

Purpose: To ascribe the serine protease inhibitor 3 (SPI-3) as an ocular acute inflammatory molecule and to clarify its producing cells in an endotoxin-induced uveitis (EIU) model., Methods: Male Wistar rats were injected with lipopolysaccharide (LPS), and the expression of SPI-3 mRNA in the ocular tissues was examined by in situ hybridization (ISH) and Northern blot analysis. A combination of ISH and immunohistochemistry (IHC) were performed to prove the colocalization of SPI-3 mRNA and either glial fibrillary acidic protein (GFAP) or OX-42. The expression of phosphorylated STAT3 (pSTAT3) was demonstrated by IHC and Western blot after LPS injection. The colocalization of SPI-3 mRNA and pSTAT3 was finally examined by the double labeling of ISH and IHC., Results: After LPS injection, the expression of SPI-3 mRNA in ocular tissues was quickly upregulated and reached a peak between 12 and 24 hours after injection. An intense mRNA signal was observed in epithelial cells of the iris and ciliary body and the innermost retinal layer. In the retina, SPI-3 mRNA was colocalized with GFAP, demonstrating that the cells expressing SPI-3 mRNA were astrocytes. After LPS treatment, SPI-3 mRNA and pSTAT3 were colocalized in retinal astrocytes, and pSTAT3 expression appeared slightly earlier than that of SPI-3 mRNA., Conclusions: Ocular inflammation induced the transient expression of SPI-3 mRNA in retinal astrocytes and epithelial cells in the iris and ciliary body, particularly during early phase of the inflammation. Simultaneously, the activation of STAT3 (phosphorylation of STAT3) occurred slightly earlier in astrocytes. This supports the previous in vitro results that SPI-3 expression is induced in a STAT3-mediated manner. SPI-3 may have some crucial roles in preventing some degenerative proteolysis, which is induced by inflammatory stimuli.

Purpose: Nitric oxide (NO) donors and cholinergic agents decrease intraocular pressure, in part because they induce a decrease in aqueous humor production. Because Na,K-adenosine triphosphatase (ATPase) is involved in aqueous humor formation, this study was conducted to investigate the hypothesis that NO and cholinomimetics regulate its activity in bovine ciliary processes., Methods: Bovine tissue slices were incubated with agonists and antagonists in a physiological buffer in vitro. Na,K-ATPase activity was determined by assaying hydrolysis of adenosine triphosphate (ATP) in suspended permeabilized tissue slices., Results: Carbachol-induced inhibition of Na,K-ATPase activity correlated with increases in cGMP. This inhibition was abolished by the muscarinic blocker atropine, the NO inhibitor N(w)-nitro-L-arginine (L-NAME) and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Sodium nitroprusside (SNP) mimicked the actions of carbachol. The SNP-induced decrease in Na,K-ATPase activity correlated with an increase in cGMP and was also abolished by ODQ. Both 8-bromo (Br)-cGMP and okadaic acid also inhibited Na,K-ATPase activity., Conclusions: Carbachol-induced inhibition of Na,K-ATPase activity involves muscarinic receptor activation. That SNP mimics and L-NAME reverses carbachol's effect on Na,K-ATPase activity suggests that the actions of carbachol are mediated by NO. Carbachol's and SNP's effects on Na,K-ATPase activity involved soluble guanylate cyclase and cGMP. Inhibition of Na,K-ATPase activity by 8-Br-cGMP and okadaic acid indicates that protein phosphorylation events may mediate SNP-induced inhibition of Na,K-ATPase activity.

Purpose: The mechanism by which the prostaglandin F2alpha-related antiglaucoma compound isopropyl unoprostone (referred to as unoprostone) reduces intraocular pressure is largely unknown. Another prostaglandin F2alpha-related compound, latanoprost, influences the activities of matrix metalloproteinases in ciliary muscle. Unoprostone ophthalmic solution is metabolized to oxidized metabolites, mainly M1 and M2, in the eye. The aim of this study was to investigate whether intraocular metabolites of unoprostone, M1 and M2, change the metalloproteinase activity in cultured monkey ciliary muscle cells., Materials and Methods: Monkey ciliary muscle cells and trabecular meshwork cells were grown separately to confluence in monolayer cell cultures. M1 (10 nM, 100 nM, or 1 microM), M2 (10 nM, 100 nM, or 1 microM), 100 nM prostaglandin F2alpha, or vehicle solutions were added to each culture medium for 48 hours. The media were then assayed to measure metalloproteinase activities quantitatively by means of substrate zymography., Results: Compared with the vehicle controls, M1, M2, and prostaglandin F2alpha significantly increased the metalloproteinase-2 activity in cultured ciliary muscle cells in a dose-dependent manner, but did not affect the metalloproteinase-2 activity in cultured trabecular meshwork cells. All experimented prostaglandins slightly increased metalloproteinase-9 activity in ciliary muscle cells, although these changes were not significant., Conclusions: The current results show that unoprostone influences the metabolism of the extracellular matrix in the ciliary muscle and that remodeling of the extracellular matrix in the ciliary muscle may be a possible mechanism by which unoprostone ophthalmic solution reduces intraocular pressure.

Purpose: The human eye is an important target tissue for steroid hormones, and glucocorticoids have been implicated in the pathogenesis of ocular disease, including glaucoma. In peripheral tissues, corticosteroid hormone action is regulated at a prereceptor level through the activity of the 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) isozymes: an oxo-reductase (11 beta-HSD1) that activates cortisol (F) from cortisone (E) and a dehydrogenase (11 beta-HSD2) that inactivates F to E. The purpose of this study was to analyze the expression and putative role of 11 beta-HSD within the human eye., Methods: Immunohistochemical and reverse transcription-polymerase chain reaction (RT-PCR) studies were performed on sections of human ocular tissues, surgical trabecular meshwork (TM) specimens and a ciliary nonpigmented epithelial (NPE) cell-line. Free F and E concentrations in aqueous humor were determined by gas chromatography-mass spectrometry (GC/MS). IOP was measured in eight male volunteers before and after oral ingestion of carbenoxolone (CBX), a known inhibitor of 11 beta-HSD., Results: 11 beta-HSD1 was expressed in the basal cells of the corneal epithelium and the NPE. 11 beta-HSD2 was restricted to the corneal endothelium. RT-PCR revealed mRNA for only the glucocorticoid receptor (GR) in the TM specimens, whereas GR, mineralocorticoid receptor and 11 beta-HSD1 mRNAs were all present in the NPE cell line. The demonstration of free F in excess of E (F/E 14:1) in the aqueous humor suggested predominant 11 beta-HSD1 activity. Compared with baseline (14.7 +/- 1.06 mm Hg, mean +/- SD), the IOP decreased significantly on both the third and seventh days of CBX ingestion (12.48 +/- 1.11 mm Hg, P < 0.0001 and 11.78 +/- 1.50 mm Hg, P < 0.0001, respectively)., Conclusions: These results suggest that the 11 beta-HSD1 isozyme may modulate steroid-regulated sodium transport across the NPE, thereby influencing IOP.

Objective: To investigate the effects of topical prostaglandin F(2 alpha)--isopropyl ester (PGF(2 alpha)-IE) administration on immunoreactivity of matrix metalloproteinases (MMPs) 1, 2, and 3 within the anterior segment tissues of monkey eyes., Methods: Eight eyes from 4 cynomolgus monkeys were evaluated. One eye from each monkey was treated with 2 mg of PGF(2 alpha)-IE twice daily for 5 days, and intraocular pressure reduction was measured. After fixation and processing, deparaffinized sections of anterior segments were immunostained using antibodies to MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), or MMP-3 (stromelysin-1). Optical density along 2 line segments overlying the iris root, ciliary muscle, and adjacent sclera and perpendicular to their long axes was measured using imaging densitometry., Results: Compared with the contralateral vehicle-treated eyes, statistically significant increases in optical density scores were observed in the iris root, ciliary muscle, and adjacent sclera for all 3 MMPs (P<.01). In these tissues, MMP-1 immunoreactivity was increased by a mean +/- SD of 89% +/- 16%, 61% +/- 8%, and 66% +/- 57%, respectively; MMP-2 immunoreactivity by 129% +/- 53%, 82% +/- 27%, and 267% +/- 210%, respectively; and MMP-3 immunoreactivity by 207% +/- 84%, 83% +/- 49%, and 726% +/- 500%, respectively., Conclusions: Treatment of monkey eyes with PGF(2 alpha)-IE induces elevation of MMP-1, MMP-2, and MMP-3 in tissues of the uveoscleral outflow pathway. These increases suggest that MMPs might play an important role in the increased uveoscleral outflow observed with topical prostaglandin treatment., Clinical Relevance: Immunoreactivity of MMPs in tissues of the monkey uveoscleral outflow pathway is increased after topical treatment with PGF(2 alpha)-IE. This response also might be involved in the intraocular pressure--lowering effect of other prostanoids used to treat glaucoma.