Immunogold localisation of laminin in normal and exfoliative iris.

Immunoelectron microscopic studies of exfoliative iris tissue (seven specimens) revealed the presence of laminin in the fibrillar component of exfoliation material. The immunogold label was uniformly distributed on the exfoliation fibres. Deposition of laminin labelled exfoliation material in the dilator muscle was a noteworthy feature, as was an apparent depletion of laminin in the basement membranes of ostensibly unaffected vessels. In control iris tissue (five enucleated eyes) laminin was identified in the basement membrane round vascular contractile cells, but not beneath the endothelium.

Prostaglandin E2 binding sites in bovine iris-ciliary body.

In the eye, prostaglandins (PGs), in particular PGE2 and PGF2 alpha, may induce vasodilation, disruption of the blood-aqueous barrier, and biphasic effects on intraocular pressure, depending on the species. The initial event leading to many of these physiologic responses is the interaction between the PG and a receptor. We have explored the specificity and selectivity of PGE2 receptors in bovine iris-ciliary body (ICB) membrane preparations. Pigment-free bovine ICB membranes were prepared by high-speed sucrose density-gradient centrifugation. Membranes were incubated with 1 nM 3H-PGE2 in the presence or absence of varying concentrations of unlabeled PGE2 or F2 alpha. Binding of 3H-PGE2 to membranes at 37 degrees C increased linearly with protein concentration, and binding reached equilibrium in 30 min. Specific PGE2 binding represented 80% of total 3H-PGE2 binding. Studies with unlabeled PGE2 or F2 alpha, as competing ligands, showed a dose-dependent inhibition of 3H-PGE2 specific binding. The IC50 for unlabeled PGE2 and F2 alpha was 3 and 379 nM, respectively, which suggests a 100-fold greater selectivity of the binding sites for PGE2 over F2 alpha. Scatchard analysis of saturation data revealed a mean Kd value of 13.3 nM with a Bmax of 156 fmoles bound/mg protein. The general linearity of our Scatchard plots tends to suggests a single class of binding sites for PGE2, although more than a single binding site could be present. These results indicate that binding sites selective for PGE2 exist in the bovine ICB.

M3-muscarinic receptor subtype predominates in the bovine iris sphincter smooth muscle and ciliary processes.

The distribution of mRNAs encoding muscarinic acetylcholine receptor (mAChR) subtypes (M1, M2, M3, and M4) was investigated in the bovine iris-ciliary body by Northern blot hybridization with subtype-specific oligonucleotide probes that were complementary to unique regions of the M1, M2, M3, and M4 mAChRs. Whole rat brain RNA, which contains all four subtypes, was employed as a positive control. Both the iris sphincter and the ciliary processes were found to contain predominantly the M3 mAChR subtype and minor amounts of the M2 subtype. Traces of the M4 subtype were detected also in the ciliary processes.

Cellular distribution of alpha B-crystallin in non-lenticular tissues.

alpha B-Crystallin is a subunit of alpha-crystallin, a major protein component of the vertebrate lens. Recently, its expression in various extra-lenticular tissues has been demonstrated by both Western and Northern blotting. In this study, the cellular distribution of alpha B-crystallin in rat organs was examined in detail using immunohistochemistry. Positive reactions were observed in lens, iris, heart, skeletal muscle (type 1 and type 2A fibers), striated muscle in skin and esophagus, Henle's loop and medullary collecting duct of the kidney, Schwann cells of peripheral nerves, glia of the central nervous system, and decidual cells of the placenta. A close correlation with markers of oxidative activity suggests that alpha B-crystallin is expressed in cells that have high levels of oxidative function.

Immunocytochemical localisation of collagens (I-V) in the human iris.

In this study we investigated the distribution of collagen types I-V in the human iris at the fine-structural level using cryoultramicrotomy and London Resin White plastic embedding. Collagen type I was shown to be present in the basement membrane of iris vessels, in contrast to type III, which was absent; both types I and III were present in the iris stroma. Collagen type IV was a major component of basement membranes of vascular cells, myoepithelial cells, fibroblasts and epithelial cells. Types II and V were absent. Both cryo and plastic embedding techniques produced closely comparable results.

Trifluorothymidine localization in the rabbit cornea by secondary ion mass spectrometry imaging microanalysis.

Imaging microanalysis by secondary ion mass spectrometry is a sensitive surface analytical technique that allows the detection and localization of elements and compounds in biological tissues. We report the detection by this approach of intracorneal trifluorothymidine following topical administration in rabbit with normal cornea. The presence of trifluorothymidine is revealed using 19F as a marker, allowing the acquisition of ultrastructural microanalytical images without using radioactive tracers.

Presence and actions of vasopressin-like peptides in the rabbit anterior uvea.

The presence of vasopressin-like immunoreactivity (VP-IR) in the rabbit eye was demonstrated by radioimmunoassay. Trigeminal nerve denervation resulted in a significant and selective decrease in the levels of VP-IR in the iris sphincter muscle and the cornea. The isolated iris sphincter muscle contracted in response to low concentrations of [Arg8]vasopressin (AVP) and related peptides. The V1 vasopressin receptor antagonist, d(CH2)5Tyr(Me)AVP, potently inhibited the contractile responses to AVP. AVP was found to induce an increase in the accumulation of inositol phosphates in the iris sphincter muscle but not in the dilator/ciliary body preparation in vitro. The present investigation demonstrates the presence of VP-IR in the rabbit eye and that this substance may be another sensory nerve-derived mediator acting on specific target sites in the anterior uvea.

Neurokinin A, neurokinin B and neuropeptide K in the rabbit iris: a study comparing different extraction methods.

Neurogenic inflammation in the rabbit eye is thought to be partly mediated by tachykinins released from trigeminal sensory nerve fibres. In the present study we have investigated the occurrence of neurokinin A (NKA), neurokinin B (NKB), neuropeptide K (NPK) and related immunoreactive components in the rabbit iris-ciliary complex using neutral and different types of acidic media for extraction, reverse phase high performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA). The immunoreactive material detected with an antiserum reacting almost equally well with NKA, NKB and NPK consisted mainly of NKA, and small amounts of NPK but almost no NKB. Acidic media seemed to be more effective than neutral media for extraction of NKA and NPK. Acid extraction yielded also an NKA-immunoreactive component which eluted immediately before NKA while neutral extracts, on the other hand, contained a component which appeared behind NKA, i.e. in the position of NKA-(3-10) and NKA-(4-10). The present results indicate that NKA but not NKB may play a role in neurogenic inflammation in the rabbit eye.

Characterization of specific binding sites for PAF in the iris and ciliary body of rabbit.

The protective effect exerted by BN 52021 a specific PAF-receptor antagonist in experimentally induced ocular inflammatory disorders led us to investigate the possible presence of specific receptors for PAF in rabbit iris and ciliary body. Two classes of PAF binding sites were found in isolated iris and ciliary process of pigmented rabbit eyes: a high affinity site Kd1 congruent to 4.9 +/- 0.47 nM, Bmax1 congruent to 3.17 +/- 0.50 pmoles/mg protein, a low affinity sites Kd2 congruent to 11.6 +/- 0.33 nM, Bmax2 congruent to 12.46 +/- 2.3 pmoles/mg protein for iris. The specific binding was not affected by lyso-PAF the biologically inactive precursor and metabolite of PAF, up to 10(-6) M; inhibition by unlabelled PAF demonstrated a biphasic curve partially antagonized by BN 52021. The present results demonstrate the presence of specific binding sites for PAF in rabbit eyes which could mediate the action of this mediator in eye inflammatory processes and explain the protective effect observed with BN 52021.

Immunohistochemical demonstration of neurotensin and tyrosine hydroxylase in iris nerves of the rabbit eye.

In previous studies we have provided evidence that intracameral administration of neurotensin (NT), an endogenous tridecapeptide, produces strong miosis in the rabbit. The presence of NT immunoreactivity was investigated in rabbit iris whole mounts by light microscopic immunohistochemistry, and its distribution in the iris compared to that, of tyrosine hydroxylase (TH). A few scattered NT-positive cell bodies were localized in the dilator muscle. Both, the NT cell bodies and processes appeared parallel to the muscle cells. Extensive branching of NT-containing cell processes was observed in connection with the sphincter muscle. These NT-positive fibers formed a dense, randomly oriented network throughout the sphincter muscle cells. The distribution of TH immunoreactivity was similar to that of NT-positive cell processes, except that no TH-positive cell bodies were detected in any of the iris structures examined. Moderate branching of TH-positive fibers was observed in the dilator and sphincter iris muscles. These findings provide neuroanatomical support for an important role of NT in pupillary physiology. Its similar topographical distribution with TH suggests that NT and dopamine may be co-localized, as it has already been described in brain.

Ocular biodistribution of clonidine after topical application with ophthalmic rods or solution.

We compared the ocular tissue and systemic blood distribution patterns of clonidine, an alpha 2-adrenergic agonist with ocular hypotensive activity, after topical application with ophthalmic rods or ophthalmic solution (eyedrops) in rabbits. We measured tissue concentrations at 0-240 minutes after administration with rods containing 5 micrograms, 10 micrograms, or 20 micrograms clonidine, or a 0.125% (62.5 micrograms) solution. The delivery efficiency of the rods was 65 - 71%. The rods and eyedrops had similar absorption and distribution patterns intraocularly and in systemic blood. Tissue concentrations of clonidine achieved were proportional to the dose delivered; peak ocular tissue concentrations were reached within 20 minutes (except for the lens). Clonidine concentrations were: tears greater than cornea greater than iris/ciliary body greater than or equal to aqueous humor greater than lens. We concluded that the ophthalmic rod offers a viable alternative to ophthalmic solution for the topical delivery of clonidine.

Association of elastin with pseudoexfoliative material: an immunoelectron microscopic study.

Using immunoelectron microscopy, the presence of elastin and tropoelastin was demonstrated in pseudoexfoliative (PSX) material in all its classical sites on the lens capsule, ciliary non-pigment epithelium, iris epithelium and stroma, and conjunctiva. Some variability in binding affinity was seen in different sites, and labelling was more often on the periphery than the center of the PSX fibers. The elastin epitope on PSX material was more sensitive to processing than the remarkably stable epitope on mature elastic fibers. Since neither elastin nor a related component of PSX fibers, elastic microfibrillar protein, is a circulating protein, both are likely to be secreted by local ocular cells. Most of these local cells are not involved in elastogenesis normally, suggesting that an abnormal stimulus or defective regulation of matrix synthesis exists in this disease.

Retinol-binding protein is synthesized in the mammalian eye.

As the chromophoric component of the visual pigment, retinol plays an essential role in vision. In the plasma, retinol is transported by retinol-binding protein (RBP) in complex with transthyretin (TTR, prealbumin). In previous work we demonstrated intraocular synthesis of TTR. To determine whether RBP is also synthesized in the eye, we performed Northern and Western blot analysis of rat eye, and detected both RBP mRNA and immunoreactive RBP. Regional Northern analysis of bovine eye localized RBP mRNA to ciliary body/iris and retina/RPE. Preliminary immunohistochemical studies revealed a widespread but heterogeneous distribution of RBP in rat eye. We postulate that ocular RBP and TTR are involved in the intraocular translocation of retinol.

[Cyclic nucleotides in experimental glaucoma]. / Tsiklicheskie nukleotidy pri éksperimental'noi glaukome.

cAMP and cGMP contents were studied in various eye tissues of rabbits with experimental glaucoma induced by chronic intravenous adrenaline administration. Cyclic nucleotide level was measured in the retina, choroid, iris and ciliary body. An increase in the tissue cAMP level was found especially in the iris and ciliary body. An increase in tissue cAMP content is explained by an enhanced beta-adrenergic regulation in the eyes of rabbits with experimental glaucoma. No consistent changes were found in cGMP content in eye tissues.

Substance P in the human iris: possible involvement in echothiophate-induced miosis in cluster headache.

Substance P-like immunoreactivity (SP-LI) was measured by radioimmunoassay in iris, choroid, and retina obtained from men after death. Although present in different amounts, SP-LI, eluting as authentic SP or SP sulfoxide in the high-performance liquid chromatography system, was found in the three ocular structures. The retina contained higher concentrations of SP-LI than the iris and choroid. The possible functional involvement of iris SP was studied in 22 episodic cluster headache (CH) patients by using the anticholinesterase agent echothiophate iodide (EI), which also induces an atropine-resistant miosis, putatively due to release of SP from trigeminal sensory neurons. In CH patients EI eye drops instilled into both eyes provoked a prolonged miosis with a more marked response in the pupil of the symptomatic eye. It is proposed that the hyperfunction of SP-containing neurons may coexist with the previously documented sympathetic hypofunction in the innervation of the symptomatic pupil of CH.

Distribution and development of substance P immunoreactive axons in the chick cornea and uvea.

A study was made of the distribution of Substance P-immunoreactive fibers in chick cornea and uvea in whole mount preparation, using the indirect immunofluorescence technique. The development of these fibers during embryogenesis was also investigated. SP-fibers were present in all chick eye structures, in the various prenatal and postnatal stages examined. Their distribution was comparable with that observed by other workers in mammals. Transformation of the iris musculature from smooth to striated, during development, is not accompanied by significant changes in SP-ergic innervation.

Ocular disposition of aminozolamide in the rabbit eye.

We have previously determined that 6-amino-2-benzothiazolesulfonamide (aminozolamide) significantly lowers IOP in rabbits and, more importantly, in ocular hypertensive human subjects. Results from in vitro experiments established that both the inhibitory activity of aminozolamide against carbonic anhydrase B as well as the penetration rate across excised rabbit corneas were equivalent to ethoxzolamide. Consequently, we have investigated the ocular disposition of aminozolamide to explain its activity when instilled topically to the eye. A constant concentration of 67.4 micrograms/ml of drug was applied for 90 min to the left eye of anesthetized rabbits. Drug and metabolite were measured in both aqueous humor and iris/ciliary body over time. The metabolite was collected and purified. Identification using mass spectroscopy, high pressure liquid chromatography (HPLC) and fluorescence scans indicated that the metabolite was 6-acetamido-2-benzothiazolesulfonamide. Relatively high levels of metabolite were identified in the cornea and iris/ciliary body but were much lower in aqueous humor. Tissue concentrations over time for the metabolite in iris/ciliary body were approximately 2-fold higher than levels of metabolite measured in aqueous humor. When compared to drug levels measured in either aqueous humor or iris/ciliary body, metabolite levels in these respective tissues were much higher. It is hypothesized that topical activity is a consequence of both metabolite retention in the iris/ciliary body as well as inhibition of 99+% of carbonic anhydrase. Both of these events must occur over a sufficient time period to effect a significant lowering of IOP.

Tissue plasminogen activator in the trabecular endothelium.

By quantitative analysis of tissue plasminogen activator (TPA) in the trabecular endothelium, corneal endothelium, and iris in the eyes of monkeys and dogs, we found significant levels of TPA activity. In a [125I]fibrin-coated well assay, the levels for the dog and monkey were, respectively: trabecular endothelium, 0.2 and 0.5; corneal endothelium, 0.8 and 0.5 IU per mg protein. The iris tissue showed high TPA activity, but its protein content could not be measured with the techniques employed. Activity in the aqueous humor was not detectable. By the ELISA technique, the values (in ng TPA/mg tissue protein) for the dog and monkey were, respectively: trabecular endothelium, 0.16 and 0.44; corneal endothelium, 0.48 and 0.92. Again, iris tissue showed high TPA activity, whereas the aqueous humor showed low activity (0.86 ng/ml). The data obtained with the two methods showed a reasonable consistency, although a direct comparison was not possible because two separate standards were used. The presence of TPA in the trabecular endothelium, corneal endothelium, and iris may be important in modulating the resistance to aqueous outflow under normal conditions as well as those of hyphema.

Affinity of ocular acid-insoluble melanin for drugs in vitro.

The authors examined spectrophotometrically the ability of drugs to bind with ocular acid-insoluble melanin. Each drug, at 5 X 10(-5) M, was incubated at 37 degrees C for 8 hr with 4 mg of melanin in 20 mM potassium phosphate buffer (pH 7.4 and 8.0) or in 20 mM acetate buffer (pH 4.8). The authors findings showed that chloroquine, thioridazine, befunolol, pindolol, daunomycin, and 5-fluorouracil bound to melanin (pH 4.8, 7.4, and 8.0). Methotrexate bound to melanin (pH 4.8 but not at 7.4 and 8.0). Pilocarpine, epinephrine, acyclovir, vincristine, and colchicine did not bind to ocular acid-insoluble melanin.