Steroid metabolism in ocular tissues of the rabbit.

The metabolism of cortisol and other steroids was studied in normal untreated rabbit iris-ciliary body and cornea as part of an investigation into the mechanism of glucocorticoid-induced glaucoma. Cortisol is readily converted to the inactive metabolite cortisone by these eye tissues indicating the presence of an 11beta-oxidoreductase system. This reaction is reversible with cortisone being converted to cortisol in the presence of appropriate cofactor. However, due to the absence of a (or as yet undetectable) cortisol-A-ring-reductase system (rate-limiting reaction) the steroid is not irreversibly metabolized to biologically inactive compounds. The 11beta-oxidoreductase system readily converts other C21-11beta-hydroxysteroids, such as corticosterone, to its appropriate C21-11-ketosteroid (11-dehydrocorticosterone). Some C21-steroids lacking the 11-hydroxyl group (11-deoxycortisol, 11-deoxycorticosterone) remain virtually unmetabolized (exception to this was found with progesterone). Evidence of a C21-steroid A-ring reductase system was found only when cortisone and progesterone were used as substrates. However, testosterone a C19 steroid was converted to clearly identifiable A-ring reduced and 17beta-and 3alpha(beta)-oxidoreduced metabolites, thus indicating the presence of testosterone A-ring reductase, 17beta-and 3alpha(beta)-oxidoreductase systems in the eye tissues studied. The presence of a steroid 5alpha(beta)-reductase for some steroids but not for cortisol indicates a distinct substrate specificity for this enzyme system in the eye tissues.     

Studies of gamma-glutamyl transpeptidase in human ocular tissues.

γ-Glutamyl transpeptidase was found to be in relatively much higher concentrations in human retina and iris than in the lens. In the lens, the enzyme was localized to a major extent in the epithelium plus capsule fraction. The lens cortex and nucleus had very little enzyme activity.γ-Glutamyl transpeptidase was purified about 200-fold to a specific activity of 897 mU per mg protein from cultured human lens epithelium. The purification steps included heating at 37° for 2 hr, deoxycholate extraction, DE-52 column chromatography, Sephadex G-200 gel filtration and affinity chromatography. The enzyme preparation was found to have a K_m of about 0·7 mm for γ-glutamyl p-nitroanilide and a pH optimum of 8·2. Magnesium had no significant effect on the enzyme activity, whereas, sodium and potassium inhibited the enzyme slightly. The competitive inhibition of γ-glutamyl transpeptidase by GSH and GSSG when γ-glutamyl p-nitroanilide was used as substrate indicates that the artificial substrate binds to the protein at the same site as the natural substrate, i.e. GSH. The enzyme was not inhibited by sulfhydryl blocking reagents up to a concentration of 1 mm indicating that the enzyme does not require the presence of sulfhydryl groups for its activity.Antibodies raised against an apparently homogeneous γ-glutamyl transpeptidase from human kidney precipitated the enzyme from ocular tissues and the enzyme purified from the cultured lens epithelial cells. This indicates that the enzyme of the ocular tissues and the kidney are of similar genetic origin.     

Effects of dorzolamide hydrochloride on ocular tissues.

We studied the intraocular pharmacokinetics of dorzolamide hydrochloride eye drops and the effect of dorzolamide on carbonic anhydrase activity and localization in ocular tissues. Carbonic anhydrase activity was detected in normal ocular tissues. The activity was inhibited in corneal endothelial cells, the ciliary body, lens epithelial cells, or the retina 1 to 8 hours after instillation of dorzolamide eye drops. In lens epithelial cells and the retina, the enzyme activity had not recovered even 10 hours after instillation of the drug. Immunostaining did not reveal any differences between the group administered dorzolamide eye drops and the control group administered a physiologically balanced solution. Time-related changes in dorzolamide concentrations in ocular tissues were measured by high-performance liquid chromatography (HPLC). In the cornea, anterior aqueous, iris, ciliary body and retina, drug concentrations increased 15 minutes after the instillation and peaked within 1 hour. These results suggest that dorzolamide immediately suppresses carbonic anhydrase activity in ocular tissues, and is rapidly distributed among the tissues of the eye when administered as eye drops.     

Effect of silicone oil on ocular tissues.

The effect of silicone oil on ocular tissues was investigated histopathologically in rabbit eyes by injecting silicone oil into the anterior chamber or the vitreous cavity after vitrectomy. The eyes were enucleated 3, 6, 9, 12 or 18 months after the injection and various tissues were observed by light and electron microscopy. Silicone oil was first observed in the retrocorneal membrane at 18 months after the injection. In the trabecular meshwork silicone oil was seen for the first time at 12 months after injection. Migrating cells engulfing silicone oil attached to the internal limiting membrane of the retina 3 months after the injection. Twelve and 18 months after the injection, silicone oil passed through the internal limiting membrane and was engulfed by Müller cells. These results indicate that attention should be paid to the effects of silicone oil on ocular tissues when it remains in the eye for a long time since, after a certain period, cells of the trabecular meshwork and retina may engulf silicone oil.     

Histologic localization of sodium fluorescein in human ocular tissues.

We studied the distribution pattern of sodium fluorescein in human eyes microscopically. The ciliary body showed early and diffuse leakage, with staining of the basement membrane of the nonpigmented ciliary epithelium, indicating movement of fluorescein from the ciliary body into the aqueous humor. After five minutes the iris stroma stained with fluorescein, probably from the aqueous humor. The retinal vessels and retinal pigment epithelium were impermeable to fluorescein. Corresponding to the background fluorescence seen in fluorescein angiography, fluorescence was present in Bruch's membrane and in the stroma of the inner one third of the choroid. Drusen stained most intensely in areas of greatest PAS positively. Early fluorescence of the optic disk was the result of intravascular perfusion of the dye. Minimal diffusion of fluorescein from the fenestrated choroidal vessels across the border tissue of Elschnig into the peripheral optic nerve bundles was observed. Late fluorescene of the optic disk was due mainly to fluorescein staining of the lamina scleralis and glial columns.     

Kallikrein and kininases in ocular tissues

The retina, choroid, ciliary body, iris and aqueous humor of swine eyes contain enzymes capable of producing (kallikrein) and inactivating (kininase I and II) kinins. The activity of the enzymes varies in different eye structures. Higher activities of kallikrein and kininase II were found in highly vascularized tissues such as retina, choroid and ciliary body. The highest activity of kininase I (carboxypeptidase N) was found in the aqueous humor. The presence of these enzymes in the eye structures suggests a possible role for them in local metabolism of vasoactive peptides.     

Freezing-thawing hysteresis. II. Investigation of human ocular tissues

The freezing-thawing behavior of cataractous eye lenses and corneas was investigated by 1H-NMR. The corneas have shown hysteresis similarly to the normal lens. No hysteresis could be demonstrated in cataractous lenses. The transparency of lens seems to be correlated with the existence of the freezing-thawing hysteresis.     

Localization of nitric oxide synthase isoforms in porcine ocular tissues.

PURPOSE: To determine by immunohistochemistry the distribution of different nitric oxide synthases (NOS) isoforms in the porcine eye. METHODS: By light microscopy (immunofluorescence), porcine ocular tissues were investigated using monoclonal antibodies against neuronal NOS (nNOS; NOS I), endothelial NOS (ecNOS; NOS III), and macrophage NOS (macNOS; NOS II). RESULTS: Specific nNOS immunoreactivity was detected along the apical cytoplasmic portions of the non-pigmented and pigmented ciliary epithelial cells, in the endothelial lining of the corneoscleral meshwork and the uveal cords of the iridocorneal angle tissue, as well as in the photoreceptor layer of the sensory retina. Immunoreactivity for ecNOS was confined to the vascular endothelium of the vessels of the conjunctiva, iris, ciliary body, retina, choroid and optic nerve. A mild immunostaining for macNOS was present in the cytoplasm of some non-pigmented ciliary epithelial cells. CONCLUSIONS: The predominant localization of nNOS in ciliary epithelial and trabecular endothelial cells suggests a possible involvement of nNOS in both the production and outflow of aqueous humor in porcine eyes.     

Pseudopodia of capillary endothelium in ocular tissues

The frequencies of pseudopodia projecting from capillaries of various parts of the eye were observed in 16 human eyes with a transmission electron microscope. The pseudopodia were found mainly projecting from the choriocapillaris and rarely seen in the retina, iris or ciliary body. The frequencies of pseudopodia from the choriocapillaris were not correlated with the localization in the fundus (macular area or peripheral fundus), age, sex, time elapsed between death or enucleation and fixation. The significance of pseudopodia from the choriocapillaris is unknown. However, there is the possibility that pseudopodia are related to choroidal neovascularization or have other physiological functions.     

Lysosomal enzymes in ocular tissues and diseases

Lysosomal enzymes are distributed widely in various ocular tissues. Among these tissues, the uvea and retina show the higher enzyme activities of acid phosphates, beta-blucuronidase, alpha-fucosidase, alpha-mannosidase, arylsulfatase, cathepsin D, cathepsin B and others. The particular role of lysosomal enzymes in the pathogenic processes of ocular diseases such as storage disease, uveitis, retinal degeneration, retinal detachment, corneal dystrophy and glaucoma is strongly suggested. The enzymes also have additional importance in ocular physiopathology.