Circadian Regulation of IOP Rhythm by Dual Pathways of Glucocorticoids and the Sympathetic Nervous System.

Purpose: Elevated IOP can cause the development of glaucoma. The circadian rhythm of IOP depends on the dynamics of the aqueous humor and is synchronized with the circadian rhythm pacemaker, that is, the suprachiasmatic nucleus. The suprachiasmatic nucleus resets peripheral clocks via sympathetic nerves or adrenal glucocorticoids. However, the detailed mechanisms underlying IOP rhythmicity remain unclear. The purpose of this study was to verify this regulatory pathway. Methods: Adrenalectomy and/or superior cervical ganglionectomy were performed in C57BL/6J mice. Their IOP rhythms were measured under light/dark cycle and constant dark conditions. Ocular administration of corticosterone or norepinephrine was also performed. Localization of adrenergic receptors, glucocorticoid receptors, and clock proteins Bmal1 and Per1 were analyzed using immunohistochemistry. Period2::luciferase rhythms in the cultured iris/ciliary bodies of adrenalectomized and/or superior cervical ganglionectomized mice were monitored to evaluate the effect of the procedures on the local clock. The IOP rhythm of retina and ciliary epithelium-specific Bmal1 knockout mice were measured to determine the significance of the local clock. Results: Adrenalectomy and superior cervical ganglionectomy disrupted IOP rhythms and the circadian clock in the iris/ciliary body cultures. Instillation of corticosterone and norepinephrine restored the IOP rhythm. ß2-Adrenergic receptors, glucocorticoid receptors, and clock proteins were strongly expressed within the nonpigmented epithelia of the ciliary body. However, tissue-specific Bmal1 knock-out mice maintained their IOP rhythm. Conclusions: These findings suggest direct driving of the IOP rhythm by the suprachiasmatic nucleus, via the dual corticosterone and norepinephrine pathway, but not the ciliary clock, which may be useful for chronotherapy of glaucoma.

Contractile markers distinguish structures of the mouse aqueous drainage tract.

PURPOSE: Structures of the aqueous humor drainage tract are contractile, although the tract is not entirely composed of muscle. We characterized the mouse aqueous drainage tract by immunolabeling contractile markers and determined whether profiling these markers within the tract distinguished its key structures of the trabecular meshwork (TM) and ciliary muscle (CM). METHODS: Enucleated eyes from pigmented C57BL/6 (n=8 mice) and albino BALB/c (n=6 mice) mice were processed for cryo- and formalin-fixed paraffin-embedded sectioning. Immunofluorescence labeling was performed for the following: (a) filamentous actin (using fluorescence-conjugated phalloidin), representing a global contractile marker; (b) α-smooth muscle actin (α-SMA), caldesmon, and calponin, representing classic smooth muscle epitopes; and (c) nonmuscle myosin heavy chain, representing a nonmuscle contractile protein. Tissue labeling was identified by confocal microscopy and analyzed quantitatively. Hematoxylin and eosin staining provided structural orientation. RESULTS: A small portion of the TM faced the anterior chamber; the rest extended posteriorly alongside Schlemm's canal (SC) within the inner sclera. Within the drainage tract, filamentous actin labeling was positive in TM and CM. α-SMA and caldesmon labeling was seen primarily along the CM, which extended from the anterior chamber angle to its posterior termination beyond the SC near the retina. Low intensity, patchy α-SMA and caldesmon labeling was seen in the TM. Myosin heavy chain immunoreactivity was primarily found in the TM and calponin was primarily observed in the CM. C57BL/6 and BALB/c comparison showed that pigment obscured fluorescence in the ciliary body. CONCLUSIONS: Our strategy of profiling contractile markers distinguished mouse aqueous drainage tract structures that were otherwise indistinguishable by hematoxylin and eosin staining. The mouse TM was seen as an intervening structure between SC, a part of the conventional drainage tract, and CM, a part of the unconventional drainage tract. Our findings provide important insights into the structural and functional organization of the mouse aqueous drainage tract and a basis for exploring the role of contractility in modulating aqueous outflow.

[The study of opportunity of aqueous humor filtration increase after nondestructive laser exposure of sclera in the site of pars plana projection (experimental study)].

Increase of scleral water permeability due to formation of porous structure after exposure of pulsed periodic radiation of erbium-glass optical fiber laser with wave length 1,56 pm was demonstrated in experimental study of cadaver human eyes in vitro and eyes of experimental animals (rabbits) in vivo. Simultaneous complex laser exposure of pars plana and ciliary processes results in summation of morphological changes that provide decrease of aqueous humor secretion, uveal drainage and extension of suprachoroid space. A base for new noninvasive technology of nondestructive laser exposure in glaucoma treatment is established.

Effect of PF-04217329 a prodrug of a selective prostaglandin EP(2) agonist on intraocular pressure in preclinical models of glaucoma.

Better control of intraocular pressure (IOP) is the most effective way to preserve visual field function in glaucomatous patients. While prostaglandin FP analogs are leading the therapeutic intervention for glaucoma, new target classes also are being identified with new lead compounds being developed for IOP reduction. One target class currently being investigated includes the prostaglandin EP receptor agonists. Recently PF-04217329 (Taprenepag isopropyl), a prodrug of CP-544326 (active acid metabolite), a potent and selective EP(2) receptor agonist, was successfully evaluated for its ocular hypotensive activity in a clinical study involving patients with primary open angle glaucoma. In the current manuscript, the preclinical attributes of CP-544326 and PF-0421329 have been described. CP-544326 was found to be a potent and selective EP(2) agonist (IC(50) = 10 nM; EC(50) = 2.8 nM) whose corneal permeability and ocular bioavailability were significantly increased when the compound was dosed as the isopropyl ester prodrug, PF-04217329. Topical ocular dosing of PF-04217329 was well tolerated in preclinical species and caused an elevation of cAMP in aqueous humor/iris-ciliary body indicative of in vivo EP(2) target receptor activation. Topical ocular dosing of PF-04217329 resulted in ocular exposure of CP-544326 at levels greater than the EC(50) for the EP(2) receptor. PF-04217329 when dosed once daily caused between 30 and 50% IOP reduction in single day studies in normotensive Dutch-belted rabbits, normotensive dogs, and laser-induced ocular hypertensive cynomolgus monkeys and 20-40% IOP reduction in multiple day studies compared to vehicle-dosed eyes. IOP reduction was sustained from 6 h through 24 h following a single topical dose. In conclusion, preclinical data generated thus far appear to support the clinical development of PF-04217329 as a novel compound for the treatment of glaucoma.

Ocular hypotensive activity of BOL-303259-X, a nitric oxide donating prostaglandin F2α agonist, in preclinical models.

The aim of the study was to investigate the ocular hypotensive activity of a nitric oxide (NO)-donating latanoprost, BOL-303259-X, following topical administration. The effect of BOL-303259-X (also known as NCX 116 and PF-3187207) on intraocular pressure (IOP) was investigated in monkeys with laser-induced ocular hypertension, dogs with naturally-occurring glaucoma and rabbits with saline-induced ocular hypertension. Latanoprost was used as reference drug. NO, downstream effector cGMP, and latanoprost acid were determined in ocular tissues following BOL-303259-X administration as an index of prostaglandin and NO-mediated activities. In primates, a maximum decrease in IOP of 31% and 35% relative to baseline was achieved with BOL-303259-X at doses of 0.036% (9 µg) and 0.12% (36 µg), respectively. In comparison, latanoprost elicited a greater response than vehicle only at 0.1% (30 µg) with a peak effect of 26%. In glaucomatous dogs, IOP decreased from baseline by 44% and 10% following BOL-303259-X (0.036%) and vehicle, respectively. Latanoprost (0.030%) lowered IOP by 27% and vehicle by 9%. Intravitreal injection of hypertonic saline in rabbits increased IOP transiently. Latanoprost did not modulate this response, whereas BOL-303259-X (0.036%) significantly blunted the hypertensive phase. Following BOL-303259-X treatment, latanoprost acid was significantly elevated in rabbit and primate cornea, iris/ciliary body and aqueous humor as was cGMP in aqueous humor. BOL-303259-X lowered IOP more effectively than latanoprost presumably as a consequence of a contribution by NO in addition to its prostaglandin activity. The compound is now in clinical development for the treatment of glaucoma and ocular hypertension.

Inhibitory effects of glucosamine on endotoxin-induced uveitis in Lewis rats.

PURPOSE: Glucosamine sulfate (GS) is a naturally occurring sugar that exerts immunosuppressive effects in vitro and in vivo. The authors investigated whether GS modulates the inflammatory reaction in endotoxin-induced uveitis (EIU) of rats and the mechanisms by which it exerts its effects. METHODS: Two-hundred micrograms of lipopolysaccharide (LPS) was injected subcutaneously into Lewis rats to induce EIU. Doses of GS (10, 100, or 1000 mg/kg) were divided into three aliquots and administered intraperitoneally 30 minutes before LPS injection, concurrently with LPS injection, and 30 minutes after LPS injection. Twenty-four hours after LPS injection, aqueous humor was collected for cell counting and measurement of protein concentration. Immunohistochemical staining of the iris-ciliary body was performed to evaluate the effects of GS on intercellular adhesion molecule (ICAM)-1 and nuclear factor (NF)-kappaB activation and to demonstrate macrophage infiltration. The effects of various doses of GS pretreatment were also examined using a mouse macrophage cell line (RAW264.7 cells) and LPS stimulation. Levels of prostaglandin (PG)-E2 and nitric oxide (NO) were determined. Expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 were measured using Western blot analysis. The effect of GS on LPS-induced NF-kappaB activation in RAW cells was also examined. RESULTS: Cell counting and analysis of protein concentration in aqueous humor revealed that GS suppressed EIU in rats treated with a high dose of GS (1000 mg/kg). Immunohistochemistry showed that treatment with GS reduced ICAM-1 expression and suppressed activation of NF-kappaB in the iris-ciliary body. The main inflammatory cells in the iris-ciliary body during EIU were macrophages. In LPS-stimulated macrophage RAW cell culture, GS inhibited the production of NO and PG-E2, the expression of iNOS and COX-2, and the activation of NF-kappaB. CONCLUSIONS: GS suppresses EIU in rats by blockading the NF-kappaB-dependent signaling pathway and the subsequent production of ICAM-1 and proinflammatory mediators. This study has extended the authors' previous observation that GS is a potentially important compound for reducing ICAM-1-mediated inflammatory effects in the eye.

Anti-inflammatory effect of angiotensin type 1 receptor antagonist on endotoxin-induced uveitis in rats.

BACKGROUND: Angiotensin II type 1 (AT1) receptor-antagonists are widely used for treatment of hypertension. Recent studies have demonstrated a protective effect of renin angiotensin system (RAS) antagonism against immune-mediated inflammatory diseases such as myocarditis, chronic allograft rejection, antiglomerular basement membrane nephritis, colitis, and arthritis. However, only a few reports have demonstrated the effect of RAS in ocular inflammatory conditions. The purpose of this study was to investigate the anti-inflammatory effect of a selective AT1 receptor antagonist, losartan, on endotoxin-induced uveitis (EIU) and compare the effect on experimental autoimmune uveoretinitis (EAU). METHODS: To induce EIU, 7-week-old Lewis rats were injected subcutaneously with 200 microg lipopolysaccharide (LPS). Losartan was administered intravenously at the same time. The aqueous humor was collected from eyes 24 h after LPS injection. The number of infiltrating cells, protein concentration, and levels of tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein-1 (MCP-1) in the aqueous humor were determined. The collected eyes were immunohistochemically stained with monoclonal antibody for activated nuclear factor (NF)-kappaB. To induce EAU, C57BL/6 mice (6-8 weeks old) were immunized with human interphotoreceptor retinoid binding protein (hIRBP)-derived peptide emulsified in complete Freund's adjuvant (CFA) and concomitantly injected with purified Bordetella pertussis toxin (PTX). Clinical severity of EAU and T cell proliferative response were analyzed. RESULTS: Losartan significantly suppressed the development of EIU. Numbers of aqueous cells of control EIU rats, those from EIU rats treated with 1 or 10 mg/kg of losartan were 75.3+/-45.6 x 10(5), 27.9+/-8.1 x 10(5), or 41.3+/-30.9 x 10(5) cells/ml respectively (p<0.01 vs control). Aqueous protein, TNF-alpha, and MCP-1 levels were also significantly decreased in a manner dependent on the amount of losartan administered (p<0.01). Treatment of EIU rats with losartan suppressed activation of NF-kappaB at the iris ciliary body. Thus, the suppressive effect of losartan on ocular inflammation in EIU appeared to result from down-regulation of NF-kappaB activation and reduction of inflammatory cytokine production. On the other hand, in the EAU model, neither the clinical score nor the antigen-specific T cell proliferative response was significantly influenced by the treatment with losartan. CONCLUSIONS: The present findings indicate that RAS may be involved in the acute inflammation of the eye, but not in T cell-dependent ocular autoimmunity. Antagonism of the RAS may be a potential prophylactic strategy for treatment of the human acute ocular inflammation.

AL-34662: a potent, selective, and efficacious ocular hypotensive serotonin-2 receptor agonist.

PURPOSE AND METHODS: The aim of this study was to determine the ocular pharmacological characteristics of AL-34662 (1-((S)-2-aminopropyl)-1H-indazole-6-ol), a new synthetic serotonin-2 (5-HT2) receptor-agonist ocular hypotensive agent. A variety of well-documented in vitro and in vivo procedures were utilized to study the pharmacological attributes of AL-34662. RESULTS: AL-34662 exhibited a high affinity for the rat and human 5-HT2 receptor (IC50=0.8-1.5 nM) and for cloned human 5-HT2A-C receptors (IC50=3-14.5 nM). AL-34662 stimulated phosphoinositide turnover in human ciliary muscle (h-CM; EC50=289+/-80 nM) and in human trabecular meshwork (h-TM; EC50=254+/-50 nM) cells. AL-34662 also mobilized intracellular Ca2+ ([Ca2+]i) in h-CM (EC50=140+/-23 nM) and h-TM (EC50=38+/-8 nM) cells, being a full agonist like 5-HT itself. AL-34662's effects in the h-CM (and h-TM) cells were potently antagonized by 5-HT2A-antagonist M-100907 (IC50=1.8+/-0.7 nM), but weakly by 5-HT2B-antagonist (RS-127445 IC50>10 microM), 5-HT2B/C- antagonist (SB-242084 IC50=2.08 microM) and 5-HT2C antagonist (RS-102221 IC50>1 microM). AL-34662 caused relatively minimal ocular discomfort and hyperemia in rabbit and guinea pig eyes. It efficaciously lowered intraocular pressure (IOP) in the conscious ocular hypertensive monkey eyes (33% at 300 microg). The (R)-enantiomer (AL-34707) and the racemate (AL-34497) were less potent and/or efficacious than AL-34662 in all of these assays. CONCLUSIONS: AL-34662 is a high-affinity 5-HT2 receptor agonist that potently mobilizes [Ca2+]i in h-CM and h-TM cells, and which efficaciously lowers IOP in conscious ocular hypertensive cynomolgus monkey eyes through a local effect with minimal side-effects.

Effect of berberine on monocyte chemotactic protein-1 and cytokine-induced neutrophil chemoattractant-1 expression in rat lipopolysaccharide-induced uveitis.

PURPOSE: The aims of this study were to examine the in vivo effects of berberine, an alkaloid isolated from some medicinal herbs, on monocyte chemotactic protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) expression in rat lipopolysaccharide (LPS)-induced uveitis. METHODS: LPS was injected intraperitoneally. Berberine was orally administered. MCP-1 mRNA and CINC-1 mRNA were measured by semiquantitative reverse-transcription polymerase chain reaction and real-time polymerase chain reaction. MCP-1 and CINC-1 protein concentration in the aqueous humor were measured by enzyme-linked immunosorbent assay. Histopathologic study was performed in the anterior ocular segments. RESULTS: Berberine dose-dependently inhibited LPS-induced MCP-1 mRNA and CINC-1 mRNA expression of the iris-ciliary body. The alkaloid inhibited chemokines, protein and cell levels in the aqueous humor in rats stimulated with LPS. On histopathologic study, the inflammatory cell infiltration was diminished by the berberine treatment. CONCLUSIONS: These findings indicate that berberine dose-dependently inhibited the expression of MCP-1 and CINC-1 induced by LPS and diminished the anterior uveitis.

Suppressive effects of astaxanthin against rat endotoxin-induced uveitis by inhibiting the NF-kappaB signaling pathway.

We investigated the effects of astaxanthin (AST), a carotenoid, on endotoxin-induced uveitis (EIU), and over the course of the disease measured the expression of inflammatory cytokines and chemokines in the presence or absence of AST. EIU was induced in male Lewis rats by footpad injection of lipopolysaccharide (LPS). The animals were randomly divided to 12 groups with eight animals in each. Immediately after the inoculation, AST (1, 10, or 100 mg kg(-1)) was injected intravenously. Aqueous humour was collected at 6, 12 and 24 hr after LPS inoculation and the number of infiltrating cells in the anterior chamber was counted. In addition, we assayed the concentration of protein, nitric oxide (NO), tumour necrosis factor-alpha (TNF-alpha) and prostaglandin E2 (PGE2). Immunohistochemical staining with a monoclonal antibody against activated NF-kappaB was performed in order to evaluate the effects of AST on NF-kappaB activation. Rats injected with AST showed a significant decrease in the number of infiltrating cells in the anterior chamber and additionally there was a significantly lower concentration of protein, NO, TNF-alpha and PGE2 in the aqueous humour. Moreover, even early stages of EIU were suppressed by injection of AST. The number of activated NF-kappaB-positive cells was lower in iris-ciliary bodies treated with 10 or 100 mg kg(-1) AST at 3 hr after LPS injection. These results suggest that AST reduces ocular inflammation in eyes with EIU by downregulating proinflammatory factors and by inhibiting the NF-kappaB-dependent signaling pathway.

Ciliochoroidal effusion induced by topical latanoprost in a patient with sturge-weber syndrome.

BACKGROUND: To report drug-induced ciliochoroidal effusion in a patient with Sturge-Weber syndrome. CASE: A 17-year-old man presented with unilateral glaucoma associated with Sturge-Weber syndrome. OBSERVATIONS: His corrected visual acuity was RE 20/20 and LE 40/60. Intraocular pressure readings by Goldmann applanation tonometry were RE 32 mm Hg and LE 12 mm Hg. Fundus examination showed marked glaucomatous disc cupping in his right eye and normal finding in his left. The patient had a port-wine stain on his right upper eyelid ipsilateral to the glaucomatous eye. Antiglaucomatous medications were begun, including topical latanoprost, with a diagnosis of juvenile onset glaucoma associated with Sturge-Weber syndrome. Ultrasound biomicroscopy showed a 360 degrees circumference ciliochoroidal effusion. Forty days after starting medication, latanoprost treatment was discontinued. Ten days later, ultrasound biomicroscopy showed a total disappearance of the ciliochoroidal effusion. CONCLUSION: Interaction of the enhanced uveoscleral outflow with latanoprost in conjunction with elevated episcleral venous pressure may have caused the congestion of the aqueous humor in the supraciliary-choroidal space, resulting in the ciliochoroidal effusion.

The mineralocorticoid receptor in rodent retina: ontogeny and molecular identity.

PURPOSE: Mineralocorticoid hormones contribute to ion-water balance in all cell types. In this study, we investigated the presence of mineralocorticoid receptors in rat and bovine ocular tissues and during retinal development. METHODS: Isolated photoreceptors and/or intact retina, retinal pigment epithelium (RPE) cells, and ciliary body were analyzed for the expression of MR (Mineralocorticoid Receptor) using the polymerase chain reaction (PCR) technique. Since aldosterone can stimulate the expression of epithelial Na+ channels (ENaC), expression of this gene in RPE was measured under basal and aldosterone-induced level. RESULTS: MR was expressed in rat photoreceptors and in the inner retina (inner nuclear layer and ganglion cell layer) even one day after birth, almost hundred percent identity was observed between rat retina and kidney MR gene products. The expression was also present in the RPE and in the ciliary body. ENaC gene was expressed in RPE and generated a predicted band at 520 bp following RT-PCR amplification which was 95% homology to that of ENaC mRNA from bovine kidney. The incubation of RPE cells in vitro with aldosterone increased the mRNA level of ENaC. CONCLUSIONS: MR expression in the mammalian retina, RPE, and ciliary body extends the potential field of action for mineralocorticoid hormones. Results on RPE cells are consistent with the idea that steroid hormones may regulate the physiology of these tissues by modulating ENaC expression. This study provides new light on the potential effect of mineralocorticoid in this area of the nervous system.

Levobetaxolol (Betaxon) and other beta-adrenergic antagonists: preclinical pharmacology, IOP-lowering activity and sites of action in human eyes.

The pharmacological characteristics of levobetaxolol, a single active isomer of betaxolol, were determined and compared with activities of other beta-adrenoceptor antagonists. Levobetaxolol (43-fold beta1-selective) exhibited a higher affinity at cloned human beta1 (Ki = 0.76 nM) than at beta2 (Ki = 32.6 nM) receptors, while dextrobetaxolol was much weaker at both receptors. Levobetaxolol potently antagonized functional activities at cloned human beta1 and beta2 receptors, and also at guinea pig atrial beta1, tracheal beta2 and rat colonic beta3 receptors (IC50s = 33.2 nM, 2970 nM and 709 nM, respectively). Thus, levobetaxolol was 89-times beta1-selective (vs beta2). Levobetaxolol (Ki = 16.4 nM) was more potent than dextrobetaxolol (Ki = 2.97 microM) at inhibiting isoproterenol-induced cAMP production in human non-pigmented ciliary epithelial cells. Levobunolol and (l)-timolol had high affinities at beta1 and beta2 receptors but were considerably less beta1-selective than levobetaxolol. Levo-, dextro- and racemic-betaxolol exhibited little or no affinity, except at sigma sites and Ca2+-channels (IC50s > 1 microM), at 89 other receptor/ligand binding sites. Levobetaxolol exhibited a micromolar affinity for L-type Ca2+-channels. In conscious ocular hypertensive cynomolgus monkeys, levobetaxolol was more potent than dextrobetaxolol, reducing intraocular pressure by 25.9+/-3.2% at a dose of 150 microg/eye (n = 15-30). Quantitative [3H]-levobetaxolol autoradiography revealed high levels of binding to human ciliary processes, iris, choroid/retina, and ciliary muscles. In conclusion, levobetaxolol is a potent, high affinity and beta1-selective IOP-lowering beta-adrenoceptor antagonist.

Acute effects of H-7 on ciliary epithelium and corneal endothelium in monkey eyes.

PURPOSE: Topical or intracameral administration of H-7 doubles outflow facility and reduces intraocular pressure in cynomolgus monkeys, by relaxing and expanding the trabecular meshwork (TM) and Schlemm's canal (SC). Since H-7 may have anti-glaucoma potential, we determined its effects on the corneal endothelium and ciliary epithelium for safety considerations. METHODS: Following topical H-7, aqueous humor flow (AHF), corneal endothelial transfer coefficient (k(a)) and anterior chamber (AC) entry of i.v. fluorescein were measured by fluorophotometry; AC aqueous protein concentration ([Protein](AC)) was determined by Lowry assay; and corneal thickness and endothelial cell density and morphology were measured by ultrasonic pachymetry and specular microscopy respectively. Following intracameral H-7, specular and/or light and electron microscopy of the corneal endothelium or ciliary epithelium were performed. RESULTS: Following unilateral topical H-7: (1) AHF and k(a) were essentially unchanged at 0.5--3.0, 3.5--6.0, and 0.5--6.0 hr, with an insignificant increase from 0.5--1.5 hr; (2) [Protein]( AC) was insignificantly increased at 1-1.5 hr but had returned to baseline by 2.5 hr; (3) entry of i.v. fluorescein into aqueous or cornea was modestly and transiently increased; (4) the central cornea thickened significantly at 1--2.5 hr, gradually returning to baseline 2.5 hr after H-7, while peripheral corneal thickness was less affected; (5) corneal endothelial cell borders became indistinct by 1 hr, but cell morphology was recovering by 3--5 hr and had completely returned to normal by 24 hr; (6) corneal endothelial cell density was unchanged at 5--24 hr. Following intracameral H-7, no significant changes were observed in corneal endothelial cell density or morphology by specular microscopy, nor in corneal endothelial or ciliary epithelial morphology by light and electron microscopy. CONCLUSIONS: A facility-effective intracameral dose of H-7 had no discernible structural effect on the corneal endothelium or ciliary epithelium. It is not yet clear whether carefully chosen topical doses of H-7 or analogues can enhance outflow facility without meaningfully affecting the cornea and ciliary processes.

Analysis of immunomodulatory activities of aqueous humor from eyes of mice with experimental autoimmune uveitis.

Aqueous humor (AqH) contains immunosuppressive factors, especially TGF-beta2, that contribute to the immune privileged status of the anterior chamber. However, this may not be true when the blood-ocular barrier is compromised by ocular inflammation. To determine the immunosuppressive status of AqH from murine eyes afflicted with experimental autoimmune uveitis, B10.A mice were immunized with interphotoreceptor retinoid-binding protein. AqH was collected from eyes of affected mice periodically after immunization and then evaluated for content of TGF-beta, proinflammatory cytokines, and the capacity to suppress anti-CD3-driven T cell proliferation. mRNA expression of selected cytokines in iris and ciliary body from inflamed eyes was analyzed by ribonuclease protection assay. We found that TGF-beta levels were significantly increased in AqH from EAU eyes on days 11, 17, and 28. AqH collected on day 11 (onset of disease) failed to suppress T cell proliferation and contained large amounts of locally produced IL-6 that antagonized TGF-beta. In contrast, AqH collected at 17 days (when ocular inflammation was progressively severe) re-expressed the ability to suppress T cell proliferation, in this case due to high levels of blood-derived TGF-beta1 and eye-derived TGF-beta2 in the absence of IL-6. Thus, during the onset of experimental autoimmune uveitis, the ocular microenvironment loses its immunosuppressive properties due to local production of IL-6. But as inflammation mounts, AqH IL-6 content falls, and the fluid reacquires sufficient TGF-beta eventually to suppress immunogenic inflammation. The paradoxical roles of IL-6 in antagonizing TGF-beta, while promoting TGF-beta accumulation during ocular inflammation, is discussed.

Intracameral anesthesia: in vitro iris and corneal uptake and washout of 1% lidocaine hydrochloride.

OBJECTIVES: To characterize the uptake, washout, and metabolism of lidocaine hydrochloride in the iris/ciliary body and cornea. METHODS: Iris/ciliary body uptake of lidocaine hydrochloride was measured by incubating human and rabbit irides in radiolabeled carbon 14-1% lidocaine hydrochloride for 2 to 60 minutes. Washout was determined by incubating the iris in 14C-1% lidocaine hydrochloride for 5 minutes and transferring the iris to a series of wells. The wells contained a common intraocular irrigating solution of essential ions, glucose, and glutathione buffered with bicarbonate (an enriched balanced salt solution [BSS PLUS]), which is similar to aqueous humor. Corneal uptake was measured by exposing the endothelial surface to 14C-1% lidocaine hydrochloride for 5 or 15 minutes. Corneal washout was performed after 5-minute exposure to 14C-1% lidocaine hydrochloride using a 2-chambered diffusion apparatus. Samples of the iris, cornea, and BSS PLUS washout solution were analyzed by high-performance liquid chromatography and liquid scintillation spectrometry. RESULTS: In vitro iris/ciliary body uptake of 14C-1% lidocaine hydrochloride follows a logarithmic curve, with 50% to 60% of maximum lidocaine hydrochloride uptake present at 5 minutes. There was no difference in uptake between human, albino rabbit, and pigmented rabbit irides. Washout of lidocaine from the iris occurs with a halflife of 8 to 9 minutes. Corneal uptake of lidocaine was greater after incubation for 15 vs. 5 minutes. The washout of lidocaine from the cornea had a half-life of 5 minutes. Results of high-performance liquid chromatography confirmed that there were no metabolites or breakdown products in the iris, cornea, or washout solution. CONCLUSIONS: Lidocaine is taken up quickly by the iris/ ciliary body and cornea and rapidly removed from these tissues after BSS PLUS washout. Irrigation during phacoemulsification seems to limit lidocaine exposure to the ocular tissues, resulting in a short duration of anesthesia. Lidocaine is not metabolized or broken down by the iris or cornea during this short period.

Association of intrinsic pICln with volume-activated Cl- current and volume regulation in a native epithelial cell.

We investigated the relationship between pICln, the volume-activated Cl- current, and volume regulation in native bovine nonpigmented ciliary epithelial (NPCE) cells. Immunofluorescence studies demonstrated the presence of pICln protein in the NPCE cells. Exposure to hypotonic solution activated a Cl- current and induced regulatory volume decrease (RVD) in freshly isolated bovine NPCE cells. Three antisense oligonucleotides complementary to human pICln mRNA were used in the experiments. The antisense oligonucleotides were taken up by the cells in a dose-dependent manner. The antisense oligonucleotides, designed to be complementary to the initiation codon region of the human pICln mRNA, "knocked down" the pICln protein immunofluorescence, delayed the activation of volume-activated Cl- current, diminished the value of the current, and reduced the ability of the cells to volume regulate. We conclude that pICln is involved in the activation pathway of the volume-activated Cl- current and RVD following hypotonic swelling.

Cannabinoid receptor CB1 mRNA is highly expressed in the rat ciliary body: implications for the antiglaucoma properties of marihuana.

We used RT-PCR to measure relative differences in cannabinoid receptor (CB) mRNAs in the rat eye, comparing CB1 or CB2 transcripts to that of the normalizing reference gene beta2 microglobulin (beta2m). Significantly higher levels of CB1 mRNA levels were found in the ciliary body (0.84+/-0.05% of beta2m) than in the iris, (0.34+/-0.04% of beta2m), retina (0.07+/-0.005% of beta2m) and choroid (0.06+/-0.005% of beta2m). CB2 mRNA was undetectable. This expression pattern supports a specific role for the CB1 receptor in controlling intraocular pressure, helping to explain the antiglaucoma property of cannabinoids.

pICln can regulate swelling-induced Cl- currents in either layer of rabbit ciliary epithelium.

Swelling-induced Cl- currents were investigated in freshly prepared non-pigmented epithelial (NPE) and pigmented epithelial (PE) cells of the rabbit ciliary body using the whole-cell patch clamp technique. Exposure of both NPE and PE cells to hypotonic stress induced Cl- currents that exhibited outward rectification and were insensitive to Ca+2. We found that swelling-induced Cl- currents in PE cell are observed shortly after isolation. The swelling-induced Cl- current showed little or no inactivation at positive membrane voltages and was sensitive to 100 microM NPPB and 100 microM DIDS. Injection of cRNA encoded rabbit pICln into Xenopus oocytes produced an outwardly rectifying Cl- current displaying features consistent with the swelling-induced Cl- current in epithelium. pICln is ubiquitous in the ciliary epithelium. It participates in the equilibration of short term tonicity alterations, a phenomenon underlying mechanisms with larger and slower amplitudes for aqueous secretion by these cells.

Functional characterization of the ocular prostaglandin f2alpha (PGF2alpha) receptor. Activation by the isoprostane, 12-iso-PGF2alpha.

Prostaglandin F2alpha (PGF2alpha) is a product of cyclooxygenase-catalyzed metabolism of arachidonic acid. Recently, PGF2alpha analogs have been hypothesized to reduce intraocular pressure via relaxation of the ciliary muscle. To investigate the molecular basis of PGF2alpha receptor (FP) activation in the eye, we cloned the FP from a human ciliary body (hcb) cDNA library. The open reading frame of the hcb-FP cDNA was identical to the uterine FP cDNA. The hcb-FP appeared to be predominantly membrane-localized, as visualized by an FP-specific peptide antibody, and coupled to inositol phosphate formation when stably expressed in HEK 293 cells. Interestingly, the hcb-FP could also be activated by the F2 isoprostane, 12-iso-PGF2alpha, in addition to its cognate ligand, PGF2alpha. 12-iso-PGF2alpha was less potent (EC50 = 5 microM) than PGF2alpha (EC50 = 10 nM) in generating inositol phosphates via the hcb-FP in HEK 293 cells. Both ligands also stimulated mitogenesis in NIH 3T3 cells. Although 12-iso-PGF2alpha caused a dose-dependent activation of the FP, it failed to activate the recombinant human prostacyclin receptor and caused only minimal activation of the thromboxane receptor isoforms stably expressed in HEK 293 cells. Four additional F2 isoprostanes, 8-iso-PGF2alpha, IPF2alpha-I, IPF2alpha-III, and 9beta,11beta-PGF2, caused trivial, or no, activation of the FP. Consistent with these observations, only PGF2alpha and 12-iso-PGF2alpha caused rapid homologous desensitization of FP and also exhibited cross-desensitization, with PGF2alpha resulting in a maximum of approximately 60% desensitization. The human FP may thus be activated specifically, by the free radical-catalyzed F2 isoprostane, 12-iso-PGF2alpha, in addition to the cyclooxygenase product, PGF2alpha. Incidental receptor activation by isoprostanes may complement the actions of PGF2alpha in clinical syndromes where oxidant stress and augmented prostaglandin biosynthesis coincide.