Identified circadian rhythm genes of ciliary epithelium with differential display.

PURPOSE: To identify differential genes expressed in the rabbit ciliary epithelium during the circadian cycle of aqueous flow. METHODS: Total RNA from ciliary epithelium of rabbits at 8 AM (light on 1 hour) and 8 PM (light off 1 hour) were compared by differential display reverse transcription-polymerase chain reaction (DD RT-PCR), using 6% denaturing polyacrylamide electrophoresis, choose differential display bands, cut and reamplify with the same primer, clone and sequence. Search the database of Genbank, prolong them with 5' RACE and 3' RACE technique then clone, sequence and search database of Genbank. RESULTS: 93 Significant differences gene expression were detected between light on and light off in the rabbit ciliary epithelium. CONCLUSION: Differential display is a powerful tool to screen differentially expressed genes in circadian rhythm of ciliary epithelium.

The role of NaKCl cotransport in blood-to-aqueous chloride fluxes across rabbit ciliary epithelium.

PURPOSE: To evaluate the role of NaKCl cotransport in short-circuit current (Isc) and chloride fluxes across rabbit ciliary epithelium mounted in a Ussing-type chamber. METHODS: Bilayered intact ciliary epithelium free of stroma was obtained after perfusion and dissection of rabbit eyes and mounted in an Ussing-type chamber. The effects of bumetanide and other drugs on Isc and transepithelial 36Cl fluxes in bicarbonate-containing Ringer's were determined. Immunoblot analysis was performed by standard techniques. RESULTS: Bumetanide (100 microM) applied to the blood (pigmented epithelium [PE]) side of the ciliary bilayer caused a dose-dependent decrease in Isc from 18.2 +/- 2.2 to 10.4 +/- 1.4 microA/cm2 (43%). Bumetanide applied to the aqueous (nonpigmented epithelium [NPE]) side of the tissue inhibited Isc by only 12%. Immunoblots of dissected NPE and PE tissue probed with an antibody to mammalian NaKCl cotransporter detected approximately 10 times more NaKCl cotransporter protein in PE than in NPE. 36Cl flux studies revealed a PE-to-NPE chloride flux of 180.3 +/- 37.2 microEq/cm2 per hour and an NPE-to-PE flux of 72.3 +/- 22.9 microEq/cm2 per hour, indicating a net PE-to-NPE flux of 108.0 +/- 31.3 microEq/cm2 per hour across rabbit ciliary epithelium. Bumetanide inhibited the PE-to-NPE chloride flux by 52% but did not inhibit the NPE-to-PE flux. Isoproterenol (10 microM) added to the PE side of the bilayer increased Isc by a dose-dependent 53%. Prior addition of bumetanide to the PE side blocked the increase due to isoproterenol by 37%. Isoproterenol (10 microM) stimulated the PE-to-NPE chloride flux by 75% but had no stimulatory effect on the NPE-to-PE chloride flux. 4,4'Diisothiocyanatostilbene-2,2'disulfonic acid (DIDS) inhibited Isc when added to either side of the bilayer but was more potent at low concentrations (<100 microM) when added to the NPE side and more potent at higher concentrations (>100 microM) when added to the PE side. Prior addition of 1 mM DIDS to the NPE side decreased isoproterenol stimulation of Isc by 56%. CONCLUSIONS: NaKCl cotransporters located primarily on the blood side of rabbit ciliary epithelium contribute to aqueous-negative Isc and to blood-to-aqueous chloride transport across the tissue in bicarbonate-containing medium. DIDS-inhibitable mechanisms, possibly including HCO3-Cl exchange and Cl channels, also play a role. Isoproterenol stimulation of Isc involves coordinate upregulation of PE-side NaKCl cotransport and an NPE-side DIDS-inhibitable mechanism(s).

Relationship between inositol 1,4,5-trisphosphate receptor isoforms and subcellular Ca2+ signaling patterns in nonpigmented ciliary epithelia.

PURPOSE: Subcellular Ca2+ signaling patterns, such as Ca2+ waves, gradients, and oscillations, are an important aspect of cell regulation, but the molecular basis for these signaling patterns is not understood. Because Ca2+ release patterns differ among isoforms of the inositol 1,4,5-trisphosphate (InsP3) receptor, the relationship between the distribution of these isoforms and subcellular Ca2+ signaling patterns in nonpigmented epithelial (NPE) cells was investigated. METHODS: The distributions of the types I, II, and III InsP3 receptors were determined in NPE cells by immunofluorescence, and subcellular Ca2+ signaling patterns in these cells were examined by confocal line scanning microscopy. RESULTS: The type I InsP3 receptor was concentrated at the basal pole of NPE cells, whereas the type III receptor was localized to the apical pole. The type II InsP3 receptor was not expressed in detectable amounts. Acetylcholine induced increases in Ca2+ that were mediated by InsP3, and these Ca2+ increases began as Ca2+ waves that were initiated at the apical pole, in the region of the type III InsP3 receptor. Acetylcholine occasionally induced sustained or repetitive Ca2+ increases that were prominent at the basal pole, in the region of the type I InsP3 receptor, but only subtle or absent apically. CONCLUSIONS: Because the type I InsP3 receptor is thought to be responsible for repetitive Ca2+ release events, and the type III InsP3 receptor instead is suited to initiate Ca2+ signals, the subcellular distribution of these two isoforms corresponds to the Ca2+ signaling patterns observed in this cell type. Differential subcellular expression of InsP3 receptor isoforms may be an important molecular mechanism by which NPE cells organize their Ca2+ signals in space and time.

Novel paracrine signaling mechanism in the ocular ciliary epithelium.

The ciliary body contains an epithelial bilayer consisting of an outer pigmented cell layer (PE) and an inner nonpigmented cell layer (NPE) responsible for aqueous humor secretion. Secretion may be mediated in part by cytosolic Ca2+ concentration ([Ca2+]i), but whether or how the two layers could coordinate their Ca2+ signals to regulate secretion is unclear. To investigate interactions between PE and NPE, we examined [Ca2+]i signaling in isolated intact ciliary epithelial bilayers using confocal microscopy. Phenylephrine selectively increased [Ca2+]i in PE and acetylcholine increased [Ca2+]i in NPE, but epinephrine increased [Ca2+]i in both layers. This increase spread from PE to NPE, and [Ca2+]i signaling across the bilayer remained coordinated during [Ca2+]i oscillations. All epinephrine-induced [Ca2+]i signaling was blocked by the alpha1-adrenergic antagonist prazosin, whereas signaling in the NPE but not PE was blocked by the beta-adrenergic antagonist propranolol, the gap junction blockers octanol and 18alpha-glycyrrhetinic acid, or the A kinase inhibitor Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate. The beta-adrenergic agonist isoproterenol failed to increase Ca2+ by itself, but isoproterenol plus phenylephrine-induced [Ca2+]i signals across the bilayer similar to those induced by epinephrine. Finally, isoproterenol increased cell-to-cell spread of lucifer yellow via gap junctions, whereas cell-to-cell spread of [Ca2+]i signals could be induced by photorelease of caged inositol 1,4,5-trisphosphate. Thus, calcium signals are coordinated in the epithelial bilayer so that adrenergic stimulation can increase [Ca2+]i in NPE, but only if NPE are primed by activation of endogenous adenylyl cyclase, whereupon they receive stimulation from adjacent PE via gap junctions. This novel interplay between endocrine and paracrine pathways may coordinate [Ca2+]i signaling across the ciliary epithelial bilayer.

Membrane carbonic anhydrase (IV) and ciliary epithelium. Carbonic anhydrase activity is present in the basolateral membranes of the non-pigmented ciliary epithelium of rabbit eyes.

Carbonic anhydrase inhibitors (CAIs) lower intraocular pressure by reducing aqueous flow. It has been thought that this pharmacologic reduction of aqueous flow is mediated by the ciliary epithelium, but it is not known whether this cellular action is effected by inhibition of the membranal (CA IV) and/or cytosolic (CA II) carbonic anhydrases of the ciliary epithelium. The isolated ciliary epithelial bilayer maintains its anatomic and functional polarity and generates a transepithelial potential difference (TEP) in an Ussing type chamber. Depletion of HCO3-, accomplished either with an HCO3(-)-free solution bathing the epithelial bilayer, or, with addition of freely permeant CAIs to HCO3(-)-containing media, (from either the PE or NPE side of the bilayer) depolarizes the preparation. Addition of CAIs to an HCO3(-)-depleted preparation has no further effect, indicating the specific action of the CAIs. The CAI, 2-p-NH2 benzenesulfonamido-1,3,4,-thiadiazole-5-SO2NH2, linked to polybutadiene maleic acid yields an impermeant polymer of 20000 Da with no loss of activity. At 45 microM this impermeant polymer caused a 60% increase in the SCC, seen only when the compound was applied to the NPE side of the bilayer. This latter result indicates an effect from inhibition of CA IV in the basolateral membranes of the NPE. Thus there are probably two different cellular actions of CAIs upon the ciliary epithelium to reduce aqueous inflow, cytoplasmic and membranal. The action of NPE basolateral membranal CA IV is probably linked to the chloride/bicarbonate exchanger.

Immunocytochemical localization of Na/K-ATPase in the isolated ciliary epithelial bilayer of the rabbit.

This study examined the localization of Na/K-ATPase in a specially isolated ciliary epithelial bilayer of the rabbit. This bilayer, harvested by a technique developed in this laboratory, consisted of pigmented epithelial (PE) and non-pigmented epithelial (NPE) cells free of stroma and with a well preserved ultrastructural morphology. Immunocytochemical localization of Na/K-ATPase was performed using goat anti-rabbit Na/K-ATPase with the biotin-streptavidin peroxidase method on fresh as well as fixed preparations. The most prominent immunostaining was found along the basolateral infoldings and interdigitations of both NPE and PE cells. This finding suggests that both ciliary epithelial layers participate in active ion transport and the production of aqueous humor.

The isolated ciliary bilayer is useful for studies of aqueous humor formation.

An intact ciliary epithelial bilayer has been isolated from the rabbit eye by perfusion, microsurgical dissection, and recovery techniques. Vital subcellular organelles and intercellular junctions of this epithelial bilayer preparation are very well preserved. The total electrical resistance of the epithelial bilayer is 350 ohms, and the transepithelial potential is 650 microV, nonpigmented epithelium side negative. The electrical resistance is reduced by 0.2 mM EGTA and the transepithelial potential reduced by 0.1 mM ouabain. Bicarbonate depletion at a constant pH of 7.4 rapidly and significantly reduces the transepithelial potential. Carbonic anhydrase inhibitors decrease transmembrane potential by as much as 30%. These morphologic and physiologic experiments authenticate the validity of this bilayered epithelial preparation for future use in detailed studies of the mechanism of aqueous humor formation.

Aqueous flare measurement with laser flare-cell meter.

The laser flare-cell meter is a newly developed instrument designed to measure anterior chamber protein concentration in vivo. There was a linear relationship between photon count measured with this instrument and the concentration of bovine serum albumin. Measurements were taken in 29 eyes of normal subjects divided into two groups. Group 1 (15 eyes) had brown or dark irises and Group 2 (14 eyes) had blue or green irises. Photon counts obtained from Groups 1 and 2 were not significantly different. Aqueous protein concentrations estimated from standard curves were 15.7 and 18.6 mg/100 ml for Groups 1 and 2, respectively. On the other hand, the background value obtained from Group 2 was about twice the value from Group 1. These results indicate that background scattering has little effect on photon count. There was a good correlation between photon counts measured with the laser flare-cell meter and the evaluation of flare made by conventional microscopic observations. It was concluded that this instrument will be useful for clinical research studies.

Alpha 2-adrenergic and VIP receptors in rabbit ciliary processes interact.

The interaction between alpha 2-adrenergic and VIP receptors has been studied by examining inhibition of VIP-stimulated cyclic AMP production by adrenergic agonists in intact, excised rabbit ciliary processes. Epinephrine, norepinephrine, isoproterenol, dopamine, and the specific alpha 2-adrenergic agonists clonidine and p-aminoclonidine exhibit dose-dependent inhibition of VIP-stimulated cyclic AMP production. I50s, clonidine (0.05 microM) = p-aminoclonidine (0.05 microM) congruent to epinephrine (0.1 microM) less than norepinephrine (2.0 microM) less than isoproterenol (15 microM) = dopamine (15 microM), are consistent with the characteristic binding affinities of these adrenergic agonists for alpha 2-adrenergic receptors. Inhibition of VIP-stimulated cyclic AMP production by clonidine, epinephrine, isoproterenol, and dopamine is blocked by yohimbine but not by prazosin. These data establish the alpha 2-adrenergic specificity of the inhibitory effects observed. We have previously shown that beta 2-adrenergic receptor-mediated stimulation of cyclic AMP production in rabbit ciliary processes is also inhibited by postjunctional alpha 2-adrenergic receptors. These studies support the idea that the catecholamines may regulate aqueous humor formation by inhibiting stimulation of cyclic AMP production via postjunctional alpha 2-adrenergic receptors in ciliary processes.

[Hypotonia bulbi and gonadotropins in myotonic dystrophy]. / Hypotonia bulbi und Gonadotropine bei myotonischer Dystrophie.

Patients with myotonic dystrophy show ocular hypotony associated with primary hypogonadism and a secondary increase in the gonadotropins FSH and LH. By examining ten patients (nine men, one woman) with myotonic dystrophy and determining serum FSH and LH concentrations by radioimmunoassay, the authors tried to evaluate whether the decrease in intraocular pressure (IOP) seen in myotonic dystrophy might be related to increase levels of gonadotropins. The IOP of the patients with myotonic dystrophy (mean 10.6 mm Hg, SD 3.9 mm Hg) was significantly lower (P less than 0.0005) than that of a randomly selected control group. In all nine male patients serum FSH concentrations (mean 41.7 mIU/ml, SD 18.9 mIU/ml, normal range 0.9-9.8 mIU/ml) and serum LH concentrations (mean 18.6 mIU/ml. SD 6.8 mIU/ml, normal range 2.2-12.0 mIU/ml) were significantly higher (FSH: P less than 0.0005, LH: P less than 0.05) than those of a control group of four normal men. The female patient's FSH was elevated (11.5 mIU/ml, normal range 1.7-8.5 mIU/ml), her LH concentration was slightly elevated (15.5 mIU/ml, normal range 2.5-15.4 mIU/ml). A statistically significant correlation was found between elevated LH concentrations and decreased IOP (P less than 0.05) but no such correlation between FSH and IOP (P less than = 0.17). Endogenous circulating gonadotropins may play an important role in regulating aqueous inflow via cyclic AMP which is produced by ciliary epithelia and causes a decrease in the rate of aqueous humor production. This theory is corroborated by humor production. This theory is corroborated by the authors' data and by experimental studies by Sears and co-workers.

Ciliary body neurilemoma recurring after 15 years.

A 57-year-old white female presented with a recurring neurilemoma (benign solitary schwannoma) of the ciliary body 15 years after the primary modified iridocyclectomy. As neurilemoma are encapsulated, successful "tumorectomy" was twice accomplished with total anatomical and functional preservation of the globe. The pathological diagnosis of spindle cell tumors of the uvea is discussed.

Treatment of elevated intraocular pressure with concurrent levobunolol and pilocarpine.

Between July 1983 and January 1986, 54 patients with open-angle glaucoma or ocular hypertension were treated for 3 months with 2% pilocarpine hydrochloride (given four times daily) and one of two beta-adrenoceptor blocking drugs, levobunolol hydrochloride (0.5% [17 patients] or 1% [19 patients]) or 0.5% timolol maleate (18 patients), given twice daily. Before entry into the study all patients had had stable intraocular pressure (IOP) with treatment with 0.5% timolol and 2% pilocarpine. Stable IOP was successfully maintained in up to 88% of the patients in the two levobunolol-pilocarpine groups and in 83% of those in the timolol-pilocarpine group. Two patients experienced adverse reactions: one, who received timolol and pilocarpine, suffered blepharoconjunctivitis, and the other, who received 1% levobunolol and pilocarpine, experienced bradycardia. The results indicate that the levobunolol-pilocarpine regimens were as safe and effective as the timolol-pilocarpine regimen in stabilizing IOP.

Interaction between alpha 2- and beta 2-adrenergic receptors in rabbit ciliary processes.

The interaction between the alpha 2- and beta 2-adrenergic receptors of ciliary processes has been studied by examining dose-response curves for adrenergic agonist stimulation of cyclic AMP production by intact, excised rabbit ciliary processes. Stimulation of cyclic AMP production by 1-isoproterenol is maximum from 0.1 to 1.0 microM; at higher concentrations stimulation decreases and approaches basal levels. Decreased cyclic AMP production at high concentrations of isoproterenol is blocked by the specific alpha 2-adrenergic antagonist, yohimbine, but not by the alpha 1-adrenergic antagonist, prazosin. Ciliary processes from animals after bilateral cervical ganglionectomy also show reduced cyclic AMP production at high concentrations of isoproterenol and this reduction is blocked by yohimbine, but not prazosin. This experiment suggests that the inhibition at high concentrations of isoproterenol is mediated by postsynaptic alpha 2-adrenergic receptors. Cyclic AMP production is relatively insensitive to epinephrine and norepinephrine, but their responses are potentiated by yohimbine. Catecholamines and clonidine, a specific alpha 2-adrenergic agonist, exhibit dose-dependent inhibition of forskolin-stimulated cyclic AMP production by ciliary processes. I50s from the dose-response curves are consistent with the characteristic binding affinities of these adrenergic agonists for alpha 2-adrenergic receptors: clonidine = epinephrine greater than norepinephrine greater than isoproterenol. Inhibition of forskolin-stimulated cyclic AMP production by clonidine is blocked by yohimbine but not by prazosin.(ABSTRACT TRUNCATED AT 250 WORDS)

Intravitreal injection of purified human chorionic gonadotropin lowers IOP in rabbits.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of two dissimilar subunits alpha and beta held together by noncovalent forces. Each subunit contains about 30% carbohydrate and is extensively crosslinked by disulfide bonds. Previous work from our laboratory with commercial preparations of hCG indicated that intravitreal injection of hCG lowered intraocular pressure (IOP). Our work has been extended by using purified hCG obtained by reverse phase high performance liquid chromatography of a commercial preparation. With a wide pore octyl silica column and a step gradient composed of dilute aqueous trifluoroacetic acid and methanol, several peaks were obtained. The major peak was shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis and amino acid analysis to contain both alpha and beta subunits. That this major peak contained intact hormone rather than a mixture of subunits was revealed by its ability to enhance the fluorescence of 8-anilino-1-naphthalene sulfonate and stimulate the release of cyclic AMP from isolated rat testes; subunits of hCG lack these properties. Physiological doses of hCG from this major peak injected intravitreally in rabbit eyes resulted in significant decreases in IOP without associated irritation when compared with contralateral control eyes.

Cervical ganglionectomy alters the circadian rhythm of intraocular pressure in New Zealand White rabbits.

A circadian rhythm of intraocular pressure in rabbits could provide a useful model for understanding the daily rhythm of intraocular pressure in humans and for studying mechanisms which regulate intraocular pressure. Our results confirm earlier work showing that New Zealand White rabbits housed in an environment with a lighting cycle of 12 hours light and 12 hours dark have a rhythm of intraocular pressure, and that this rhythm persists in constant dark. We show further that the cycle of light and dark is the zeitgeber for entrainment of the rhythm of intraocular pressure, and therefore persistence of this rhythm in constant dark establishes it as a circadian rhythm. Cervical ganglionectomy demonstrated that intact sympathetic innervation to the eye is required for maintenance of the normal circadian rhythm of intraocular pressure in rabbits. Intraocular pressure in sympathectomized eyes is no different from control eyes during the light, but is significantly reduced during the dark.