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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.

Beta adrenergic binding sites were localized and characterized in the human eye by means of "in vitro" autoradiography, using [125I] (-) iodocyanopindolol (125ICYP) as radioligand. Binding sites were visualized by apposition of isotope sensitive film to slide mounted eye sections. Receptor sites were present in the extraocular muscles, in the conjunctiva, in the epithelium and endothelium of the cornea, in the trabeculum and in the ciliary muscle. They were also present in the lens epithelium and in the retina. The pigmented ocular structures were heavily labelled but the binding was nonspecific. Characterization of these binding sites was achieved by testing the ability of selective adrenergic compounds to displace 125ICYP binding. These studies suggested that the majority of adrenergic binding sites in nonpigmented structures of human eye were of a beta2 type.

A cartilage matrix glycoprotein (CMGP), previously identified in human and bovine vitreous, now has been found in the vitreous body of rabbits aged 1-22 months by immunohistochemical techniques. Epithelial cells of the inner layer of the ciliary epithelium contain material that has immunologic cross-reactivity with a specific antibody to CMGP. These cells also secrete glycoproteins, as determined by autoradiography after intravitreal injection of [3H]fucose. Approximately 14 bands, representing intrinsic glycoproteins containing fucose residues, can be identified in fluorograms of SDS-polyacrylamide gels of vitreous bodies from 6- and 22-month-old rabbits. Fluorograms of gels of samples of vitreous and ciliary bodies from several time points after intravitreal injection of [3H]fucose reveal at least seven comigrating protein bands and also demonstrate turnover of the labeled ciliary body glycoproteins. These results suggest that the inner layer of the ciliary epithelium is the source of the glycoproteins of the vitreous body and that these glycoproteins undergo turnover, probably throughout the entire life of the animals.

Guanine nucleotide-binding proteins (G proteins) are a family of receptor-coupled signal-transducing proteins that regulate a variety of second-messenger systems and ion channels. The complement of G proteins in SV40-transformed pigmented and nonpigmented ciliary epithelial cells was determined by Western blot analysis utilizing peptide and holoprotein derived antisera to known G protein alpha and beta subunits and cholera toxin catalyzed ADP-ribosylation. The complement of alpha subunits found in both SV40-transformed NPE and PE cells includes Gs alpha and all three members of the Gi alpha family. Neither cell type contains Go alpha or Gz alpha. Both cell lines contain beta 35 and beta 36. Future studies will examine the functional involvement of these G proteins in the regulation of aqueous humor stimulus-secretion coupling.

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.

Cells containing smooth muscle myosin were localized in the human aqueous outflow pathway by immunohistochemical techniques. In the majority of eyes, immunoreactive cells were observed adjacent to the collector channels and slightly distal to the outer wall of Schlemm's canal. In a few eyes, smooth muscle myosin was localized to cells in the juxtacanalicular tissue and the trabecular meshwork. The immunoreactive cells from these regions may be true smooth muscle cells or pericytes, which can contain smooth muscle myosin. No obvious differences were observed in the pattern of distribution of smooth muscle myosin-containing cells in a comparison of age groups. In the majority of eyes, we observed an apparent direct insertion of the longitudinal portion of the ciliary muscle in the corneoscleral meshwork far internal to the scleral spur.

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.

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.

DARPP-32, a phosphoprotein enriched in dopaminoceptive brain neurons containing the D-1 receptor subtype, probably functions as an intracellular third messenger to mediate some of the physiological effects of dopamine at the D-1 receptor. By immunohistochemistry in rat, cat, Rhesus monkey, and human, we have localized DARPP-32 to the non-pigmented epithelium of the ciliary body, the innermost layer of the bi-layered epithelium responsible for secretion of aqueous humor into the eye. The immunoreactive protein in rat ciliary body, identified by immunolabeling of a ciliary body extract separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, is indistinguishable from DARPP-32 derived from rat caudatoputamen. By analogy with brain, we propose that DARPP-32 may act as a third messenger in the ciliary epithelium, probably through a dopaminergic mechanism.

Cholinergic receptors were studied in membranes prepared from rabbit cornea, iris-ciliary body, and retina, using 3H-quinuclidinyl benzilate (3H-QNB) to identify muscarinic receptors and 125I-alpha-bungarotoxin (125I-BGT) to identify nicotinic receptors. Muscarinic cholinergic receptors were not found in the cornea. As a positive control, muscarinic cholinergic receptors were characterized in preparations of the iris-ciliary body. Specific binding of 3H-QNB to iris-ciliary body membrane preparations was saturable, with a Kd of 1.3 nM QNB. Specificity of the assay for muscarinic receptors was confirmed by the relative abilities of the following compounds to displace 3H-QNB: atropine greater than pilocarpine greater than hexamethonium. Nicotinic cholinergic receptors were not found in the cornea. As a positive control, nicotinic cholinergic receptors were characterized in preparations of the retina. Specific binding of 1252-BGT to retinal membrane preparations was saturable with both high and low affinity receptors (Kd values of 1.0 nM and 93 nM BGT, respectively). Specificity of the assay for nicotinic receptors was confirmed by the relative abilities of the following compounds to prevent 125I-BGT binding: curare greater than or equal to nicotine greater than hexamethonium greater than atropine. The lack of cholinergic receptors in the cornea, which has high levels of acetylcholine and related enzymes, suggests either an extraordinary use or a lack of function for acetylcholine in this tissue.

Evidence is presented that serotonin acts as a neurotransmitter in the cornea of the adult rabbit. Serotonin was localized to granules in a sparse population of subepithelial corneal nerves by an electron microscopic histochemical procedure. Significant endogenous levels of serotonin and its principal metabolite, 5-hydroxyindoleacetic acid, were detected in the central cornea by a fluorometric assay. Exogenous serotonin stimulated ion transport by corneal epithelium. This effect was potentiated by monoamine oxidase inhibition and was unaffected by an alpha-adrenergic receptor antagonist. Serotonin-stimulated ion transport was inhibited by the specific antagonist, methysergide, and by the replacement of Cl- with an impermeable anion. In tracer experiments, the serotonin-stimulated ion transport was shown to be caused by increased epithelial Cl- secretion. The serotonin response was partially inhibited by the beta-adrenergic antagonist, timolol. In a companion article, assay of corneal cyclic AMP showed stimulation of cyclic AMP synthesis by serotonin, inhibition by the specific antagonist, lysergic acid diethylamide, and potentiation by monoamine oxidase inhibition. We postulate that specific serotonergic receptors are present in the corneal epithelium and that activation of these receptors by serotonin released from serotonergic neurons increases the level of cyclic AMP, which stimulates active Cl- secretion by the corneal epithelium.

After removal of the supper cervical sympathetic ganglion in cats followed by increase of the intraocular pressure from 20 to 32 mm Hg, the outflow of intraocular fluid was sharply enhanced due to lowering tone of tissues of the eye drainage apparatus in the desympathised eye. Noradrenaline was found to reduce sharply by the 4th-5th day in the desympathised eye's tissues due to degenerative processes whereas accumulation of noradrenaline by the 6th-8th day occurred on account of humoral factors.

Cathepsin B in the bovine ciliary body and iris was studied biochemically using alpha-N-benzoyl-D,L-arginine-2-naphthylamide as substrate. The enzyme was purified to 210-fold from the autolyzed extract. The partially purified enzyme had a pH optimum at 6.0 and a molecular weight of 27,000. The apparent Km value for the substrate was 1.6 mM. The enzyme was activated by disodium ethylenediamine tetraacetate, cysteine, and dithiothreitol. The enzyme activity was inhibited strongly by leupeptin and partially by hyaluronate and chondroitin sulfate A.

Human ciliary body epithelial cells have been maintained in vitro and have been partially characterized by the determination of growth rate, morphology, and ultrastructural parameters. The dissection technique employed allows the separation of pure ciliary body epithelium with a predominance of cells being from the nonpigmented layer. Growth curves indicate this cell population follows a prolonged rate of growth compared to other primary cell cultures. Loss of pigment granules noted by light microscopy were documented by morphometric analysis of electron micrographs. Thirty-two percent of the cultures attempted were successful. Maintenance of these cells in vitro may provide a means for studying their enzyme systems, growth factors, reactions to various stimuli, and the effects of this cell population on other intraocular tissues.

Flow cytometry was used to evaluate ploidy and tumour cycle kinetics in fresh tissue samples obtained from 19 uveal melanomas. The results were compared with other parameters including, histological cell type, tumour size and anatomical location. Three tumours (15.8%) were aneuploid (two mixed cell, one epithelioid cell). Cell turnover was estimated in the 16 diploid tumours by summating the total percentage of cells in S and G2/M phases. We found the mean percentage of cells in G2/M/S to be 5.96% (range 2.2-9.8%). Spindle cell neoplasms appeared to have lower cell turnover rates (4.5 +/- 1.2%) than epithelioid cell turnover (8.4 +/- 1.2%). There was no correlation between cell turnover and either tumour size or anatomical location.

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.

Angiotensin II (A2) is a vasoconstrictor generated by the renin-angiotensin system. A2 appears to act also as an angiogenic factor. Recent evidence suggests that renin is synthesized at many tissue sites and may generate A2 locally. Local A2 may have important functions in the normal and diseased eye. We examined eight human eyes by immunostaining with an antibody to prorenin, the biosynthetic precursor of renin. In all eyes, prorenin staining was extensive in the pars plicata of the ciliary body suggesting that the ciliary body synthesizes renin and this renin may be part of an ocular A2 generating system.

The ATP content in the rabbit eye tissues increases from birth till the time of sight appearance (12-15th day after birth). The ADP and AMP content did not increase during the period of sight appearance. After this period, the ATP content decreased down to the level of newborn tissues. The ADP and AMP content increased in the eye tissues of 2 months old and adult rabbits.

Substance P (SP) levels in rabbit iris-ciliary body preparations were found to be significantly elevated 30 days after removal of the superior cervical ganglion when compared with the contralateral unoperated side. These elevated levels were still apparent after two and three months. Immunohistochemical studies, using a monoclonal antibody to SP, revealed an increase in the density of SP-immunoreactive nerve fibres in the region of the iris dilator muscle and in the ciliary processes one to four months after sympathectomy, although the density of these fibres in other regions of the iris was not altered.

The content of the fibronectin, an extracellular glycoprotein in the drainage outflow system of human eyes was determined by the indirect immunoperoxidase staining technique. The degree of fibronectin accumulation in ocular tissues was evaluated by quantitative morphometric analysis. It was shown that the fibronectin level was elevated in ageing. Increased deposit of fibronectin in trabecular tissues, mainly, in the inner wall of Schlemm's canal and juxta-canalicular zone was demonstrated along with ageing. Comparison of fibronectin accumulation in glaucoma and ageing support the idea that ageing is a risk factor of glaucoma.

The distribution of cytokeratin and vimentin in guinea pig tissues as seen by immunohistochemistry using monoclonal antibodies is described and a similar distribution pattern of coexpression of cytokeratin and vimentin in various cell types as compared to human tissues were found. The possible explanations for the unusual coexpression of both types of intermediate filaments in normal cells are discussed.

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.

By measurement of the specific binding of 3H-dihydroalprenolol, the densities of beta-adrenergic receptors on membranes prepared from homogenized corneas and iris--ciliary bodies of rabbits were studied. Sympathetic denervation, as a result of subconjunctival treatment with 6-hydroxydopamine, causes an increase in the density of beta-adrenergic receptors in membranes prepared from the ipsilateral iris--ciliary body but not the cornea. Topical treatment with epinephrine for 5 days causes a decrease in the density of beta-adrenergic receptors in membranes prepared from cornea and iris-ciliary body, whereas similar treatment with timolol causes an increase in the density of beta-adrenergic receptors. In the cornea, the decrease in receptor density that occurs following in vivo treatment with epinephrine is associated with a decreased ability to synthesize cyclic AMP, whereas the increase in receptor density that occurs following in vivo treatment with timolol is not associated with an altered ability to synthesize cyclic AMP. Our results indicate that the density of beta-adrenergic receptors in the anterior segment of the eye is inversely related to the level of adrenergic stimulation to the tissue but that the ability of a tissue to synthesize cyclic AMP does not necessarily parallel the change in receptor density.

Since intravenously injected Evans blue binds irreversibly to serum albumin, its distribution reflects albumin exchange between the intravascular and extravascular tissue compartments. In histologic specimens examined by fluorescence, microscopy, extravasated Evans blue--albumin complex was identified within the ciliary body and trabecular meshwork of normal monkey eyes. In eyes fixed by intra-arterial perfusion of fixative, no dye was identified in the choroid, retina, or optic nerve. With immersion fixation, however, some extravasation was seen in the choroid and adjacent optic nerve. In some specimens, the optic nerve was stained not only with material apparently leaking from the choroid but also from a breakdown of the blood-brain barrier in the major disc vasculature during the interval before fixative penetrates into the tissue. Perfusion fixation must be used to avoid this artifact, and freezing techniques would be even better.

The present study involves the intracardial injection of iodinated ovine prolactin into albino rats and the autoradiographic demonstration of patterns of isotopic incorporation into ocular tissues, including the retina, choroid coat and ciliary body. Control procedures include the utilization of competitive hormonal binding, a comparison of the radioactive pattern in eyes after the injection of iodinated beef serum albumen and an autoradiographic evaluation of the thyroid glands of all groups. The competitive uptake of iodinated prolactin into ocular tissues also was examined by liquid scintillation spectrometry. In normal rats, radioactivity was localized over the cells of the choroid coat and ciliary body of all groups receiving iodinated prolactin. The inner segments of the photoreceptors were radioactive in rats at 15 min after injection, but were unlabeled at only 5 min after injection.

Eyes from adult rodents, rabbits and humans were fixed in formalin, embedded in paraffin and incubated with a horseradish peroxidase-conjugated lectin from Ulex europaeus to localize vitreous-body and zonular glycoconjugates. Rodent eyes had reaction product for peroxidase activity in fibrous structures in the posterior chamber, vitreous base and vitreous cortex. The zonules and the internal limiting membrane region of the retina also were stained. Rabbit eyes had more stained fibrous material in the vitreous base than rodent eyes and the attachment region of the zonules on the lens capsule, the anterior hyaloid membrane and tracts in the vitreous cortex were more heavily stained in rabbits. There was heavy staining of the thick vitreous base in the human eyes as well as staining of zonules, anterior hyaloid membrane and vitreous cortex. The localization of this lectin may be specific for fucose in glycoproteins or other glycoconjugates, although this was not demonstrated here. However, the location of lectin binding sites correlates well with known sites of uptake of tritiated fucose and tritiated glucosamine in rabbit eyes. Eyes from the larger species studied had more lectin-binding glycoconjugates in fibrous structures in the vitreous body than those from smaller species. The amount of glycoconjugate identified in some of the lectin-binding sites may be related to some extent to the degree of stress incident upon those sites.

The level of ascorbate in the aqueous humor and surrounding intraocular structures in enucleated arterially perfused rabbit eye was investigated. The enucleated eye preparation was shown to be capable of secreting ascorbate from the perfusate into the aqueous humor. Ascorbate in the iris, ciliary body and cornea was released into the aqueous humor when the eye was perfused with ascorbate-free solution. Failure to obtain aqueous flow rates from the decay of ascorbate in the anterior chamber was due to the contribution of ascorbate from these ocular tissues during the perfusion. Histochemically, ascorbate was localized in the pigmented epithelial layer in the valleys between the ciliary processes and the pars plana of the ciliary body and in the iris. In the cornea, distinct localization of ascorbate was observed in the endothelium and basal cell layer of the epithelium.

Homozygous 020/A mutant mice bearing the rds gene for slow inherited retinal degeneration have been observed to develop normal photoreceptor inner segments connecting cilia and synaptic contacts but fail to form outer segments. Their retinas are responsive to light, however. In order to assess the sources of these physiological responses we investigated the distribution of opsin in photoreceptors by means of immunoelectron microscopy. Opsin was detected in the inner segment plasma membrane and the distal ciliary plasma membrane. Antibody also bound to lamellar and vesicular membranes in the interphotoreceptor space and, in a small fraction of the photoreceptors, to membranes projecting from the distal cilium. These membranes may represent abortive formation of rod discs in this form of retinal degeneration. Failure to form an organized outer segment may contribute to the persistence of opsin in the inner segment plasma membranes of adult mutant mice.

A comparison was made between the ocular penetration of topically applied 14C-atenolol, 14C-timolol, 14C-propranolol and 3H-metoprolol by means of liquid scintillation counting. Only one eye was treated, the fellow eye served as a control. Blood plasma levels were measured as well. We could detect a relationship between the ocular penetration and the degree of lipophilicity of the drugs used, as was to be expected. Drugs with a higher degree of lipophilicity penetrated more readily into the eye, whereas they also achieved higher blood plasma levels. Relatively high concentrations of atenolol were found in the nicitating membrane, thus reflecting its poor ocular penetration. The concentrations detected in the untreated eye were low and probably cannot explain the marked reduction in intraocular pressure observed after unilateral instillation of active beta-adrenergic blocking drugs, as for instance timolol. Our findings suggest that the ocular penetration of beta-adrenergic blocking agents on topical application only plays a minor part in their ocular hypotensive effect.

Specific, high affinity atrial natriuretic factor (ANF) binding sites were identified and localized by autoradiographic techniques in peripheral tissues of the guinea pig, rat, and human. In the guinea pig kidney, high concentrations of ANF binding sites were located in the glomerular apparatus, outer medulla, and small renal arteries. Other peripheral tissues containing ANF binding sites included the zona glomerulosa of the adrenal cortex, the smooth muscle layer of the aorta and gallbladder, the lung parenchyma, the posterior lobe of the pituitary, the ciliary body of the eye, and the leptomeninges and choroid plexus of the brain. The distribution of ANF binding sites in the rat and human kidney was nearly identical to those seen in the guinea pig kidney; high concentrations were present in the glomerular apparatus, outer medulla, and small renal arteries. These results are consistent with earlier physiological and pharmacological studies that suggested that ANF plays a functional role in the regulation of extracellular fluid volume and blood pressure. There appears to be little species variation in the location and concentration of renal ANF binding sites, suggesting that, at least in the kidney, the results in experimental animals are relevant to the actions of ANF in humans. The finding that ANF binding sites were stable and present in high concentrations in human postmortem kidneys further suggests that these tissues may be amenable to testing for the involvement of ANF receptor dysfunction in diseases such as hypertension and congestive heart failure.

A solid adenoma of the pigmented ciliary epithelium was removed by 9.0 X 6.0 mm "block-excision" (postoperative visual acuity 1.0). The black lesion with a velvety surface consists of very large cells packed with melanin granules of abnormal and variable density, structure and size. Striking intracellular vacuoles contain acid mucopolysaccharides, particularly sialomucins. The literature on tumors of the pigmented ciliary epithelium is reviewed. Criteria for the separation of adenomas for adeno- "carcinomas" (8 cases, all without metastases) from reactive hyperplasia (6 cases) are proposed. The histologic, histochemical and electronmicroscopic findings of our tumor show striking similarities to those of an adenoma of the retinal pigment epithelium.

A previous investigation had indicated that pilocarpine delivered to the eye by means of a hydrophilic contact lens provided better control of the intraocular pressure over a longer period and with a smaller concentration of pilocarpine. To clarify the situation further, an animal experiment was undertaken with radioactive labelled pilocarpine. Three situations were compared: pilocarpine administered by a contact lens, by drops, and by subconjunctival injection. The results indicated that the contact lens gave a greater concentration of the pilocarpine in the iris and ciliary body than either the drops or the subconjunctival pilocarpine. A high concentration of pilocarpine was achieved in the conjunctiva with drop administration and was comparable to that of subconjunctival administration.

Selective autonomic denervations of the iris have been used to study the possible redistribution of adrenergic markers within adult nerve fiber systems and to reveal the cellular origin of a nonsympathetic fiber plexus induced to express such markers. The presence and distribution of fibers showing neuropeptide Y (NPY)- and tyrosine hydroxylase (TH)-like immunoreactivity was studied in the rat iris using stretch-prepared whole mounts. Normal irides contained a dense regular network of NPY-positive varicose fibers. Such fibers were regularly seen innervating blood vessels. The choroid membrane had a high number of fluorescent fibers. A similar, although slightly denser TH-positive fiber system was visualized in the iris. One or 2 days after surgical removal of the superior cervical ganglion, almost all NPY- and TH-positive fibers had disappeared, suggesting that most, if not all, NPY-positive fibers in the iris originate in the superior cervical ganglion. In irides from long-term sympathectomized animals, a high number of TH- and NPY-immunoreactive fibers had reappeared, while such irides were devoid of catecholamine-containing fibers, as evidenced by Falck-Hillarp histochemistry. The appearance of TH- and NPY-positive fibers in sympathetically denervated irides was clearly time dependent. The distribution of fluorescent fibers in irides from intact and sympathectomized animals showed obvious dissimilarities such as a lower fluorescence intensity and fewer varicose fibers in denervated irides. Furthermore, in irides from sympathectomized rats, TH- and NPY-positive fibers were not associated with blood vessels. Unilateral removal of the parasympathetic ciliary ganglion, which supplies the iris with cholinergic fibers, 3 days prior to sacrifice in animals bilaterally sympathectomized 1 month earlier, led to a drastic reduction in numbers of TH- and NPY-positive iris fibers on the ciliarectomized/sympathectomized side as compared to the sympathectomized-alone side. The present experiments thus suggest that adult cholinergic neurons in vivo are capable of expressing adrenergic characteristics under experimental conditions.

The authors have determined the acetylcholine (ACh) content in bovine ciliary bodies. One of the ciliary bodies was subjected in vivo to the influence of an aqueous solution of pilocarpine HCl 3 p. 100 which was instilled in the conjunctival cul-de-sac (ten times at a dose of three drops every three minutes). The contralateral ciliary body of the same animal served as a control for the ACh concentration. Enucleation was performed one hour after the last instillation. The average concentration of ACh estimated in 13 control ciliary bodies was 0,40 mug of ACh, and in 13 pilocarpinised eyes 0,50 mug of ACh (for 150 mg of tissue). The questions concerning the inactivation of pilocarpine and the source of augmentation of cholinergic tone in the pilocarpinised eyes have been discussed in previous communications.

Levels of free amino acids were determined quantitatively in whole ocular tissues of the rat eye with aid of a sensitive amino acid analyzer. The tissues studied were the retina, vitreous, lens, iris-ciliary body, and cornea. The retina and lens contained a more concentrated free amino acid pool than other tissues. The neuroactive amino acids taurine. GABA, glutamic acid, aspartic acid, and glycine were clearly enriched in the retina. Taurine was the most abundant amino acid in all five tissues studied, and its high concentration in non-neural tissues, especially the lens, suggests that it must have other functions as well as neurotransmitter ones in the rat eye.

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.

Dexamethasone binding to both rabbit iris-ciliary body and rabbit liver glucocorticoid receptors was compared by means of a batch assay in which the binding components were adsorbed to hydroxylapatite. The results showed that homogenates of both the rabbit iris-ciliary body and the rabbit liver contained a single class of high-affinity receptors. Binding affinity was virtually identical for the two receptors. A comparison of total binding sites in homogenates of the iris-ciliary body and the liver revealed that the iris-ciliary body glucocorticoid receptor was present in nearly twice the concentration of the hepatic receptor.

Vitreous body fibrils and zonula fibers are analyzed by disc-electrophoresis. In both tissues the presence of collagen is established. The disc-electrophoresis patterns of vitreous body fibrils and zonula fibers show that a different type of collagen is present in each tissue. The absence of the alpha 2-chain indicates that the tissues investigated contain no type I collagen but types consisting of three identical alpha-chains.