Chromatographic characterization of vasoactive intestinal polypeptide in guinea pig and rhesus monkey eyes.

Acid extracts of guinea pig and rhesus monkey anterior uvea, choroid and retina contain immunoreactive VIP. By reversed phase high performance liquid chromatography, the immunoreactive material from these ocular tissues elutes at a position similar to synthetic porcine VIP. Only in the guinea pig anterior uvea is a second smaller peak detected. By size exclusion high performance liquid chromatography, the peptide in the monkey anterior uvea, choroid and retina elutes in the identical position as the synthetic porcine VIP standard with an apparent molecular weight of 3450 daltons. We conclude that a single form of VIP, chromatographically similar to the porcine standard, is the predominant form of the peptide in the eye of guinea pig and rhesus monkey.

Immunohistochemical localization of peroxidative enzymes in ocular tissue.

Localization of the peroxidative enzymes catalase and glutathione peroxidase in rabbit ocular tissue was investigated by immunohistochemical methods. We raised antisera to both enzymes in rabbits using commercially available purified enzymes. Immunoreactive catalase and glutathione peroxidase were found in the corneal epithelium and endothelium, the choroid, the inner segment of photoreceptors, and the retinal pigmented epithelium.

Localization of gamma glutamyl transpeptidase in bovine retina.

Gamma-glutamyl transpeptidase (GGTP) is a membrane bound enzyme which has an important role in regulation of glutathione and glutamate in the retina. We have used histochemical and colorimetric enzyme assays to localize GGTP in the bovine retina and choroid. Our results demonstrate that (i) GGTP is present in retinal microvessels but not choroidal microvessels. (ii) Retinal microvascular endothelium loses the ability to express GGTP in cultured cells. (iii) GGTP is present in Muller cells. (iv) Isolated and purified rod outer segments contain high levels of GGTP. (v) Retinal pigment epithelial cells (RPE) in vivo and in culture contain GGTP. The findings of this study lend support to the concept that GGTP may be a biochemical marker for cellular systems which are part of specialized diffusion barriers.

Binding and transport of transthyretin-gold by the endothelium of the rat choriocapillaris.

The photoreceptors of the neural retina require retinol for synthesis of rhodopsin. In the plasma, retinol is bound to retinol binding protein which is carried by transthyretin (TTR; formerly called prealbumin). It is unknown whether, or how, retinol carrier proteins cross the endothelium of the choriocapillaris, the blood supply to the outer neural retina. This was examined in the present study with TTR-gold probes perfused into rats and localized by electron microscopic techniques. TTR-gold, often in clusters, was localized to diaphragmed fenestrae, parajunctional areas, coated pits, transendothelial channels, multivesicular bodies, and to vesicles close to the Golgi apparatus. The probe was also identified at the luminal and abluminal fronts and the interior of transendothelial channels in an apparent sequence of transit. TTR-gold was also found in a series of interconnected vesicles adjacent to the abluminal side of the endothelium. Localizations were not seen when rat albumin fraction V was substituted for TTR and when the rats were perfused with Pronase E before labeling with TTR-gold. These observations indicate that binding and receptor mediated-like transport of TTR by the endothelium of the choriocapillaris is present. This is similar to the processing of heparin-gold by this endothelium.

Analysis of proteins in developing rd (retinal degeneration) chick retina using two-dimensional gel electrophoresis.

The rd (retinal degeneration) chick possesses an autosomal recessive mutation which results in behavioral and electrophysiological blindness at hatch. Using the technique of two-dimensional gel electrophoresis, we have identified two groups of proteins whose expression in the retina/pigment epithelium/choroid (RET/PE/CH) of +/+, +/rd and rd/rd chicks is related to genotype. The two proteins within Group 1 had an apparent mass (Mr) of 63 kDa and isoelectric points (pI) of 6.48 and 6.55. The four proteins in Group 2 had an apparent Mr of 98 kDa and pI values ranging from 6.08 to 6.25. Quantities of the Group 1 proteins in RET/PE/CH of each of the three types of animals were found to be related to genotype; the amounts of each did not change with development. The expression of the Group 2 proteins in PET/PE/CH was found to change during retinal development. Proteins P98-6.08 (Mr-pI) and P98-6.13 were found in all rd/rd and +/rd RET/PE/CH and in most +/+ embryonic tissues. Proteins P98-6.19 and P98-6.25, which were present in the majority of +/+ embryonic and in all +/+ E21 (hatch RET/PE/CH, did not appear in +/rd tissues until hatch. P98-6.19 and P98-6.25 were never observed in rd/rd RET/PE/CH.

The autofluorescent products of lipid peroxidation may not be lipofuscin-like.

The fluorescent molecules of cellular age pigment granules (lipofuscin) are commonly thought to be end products of membrane lipid autoxidation. Lipofuscin fluorophores of the retinal pigment epithelium (RPE) appear to be derived from photoreceptor outer segment membranes. Experiments were therefore conducted to determine whether the in vitro oxidation of retinal homogenates would generate fluorophores similar to the naturally occurring lipofuscin fluorophores of the RPE. Neural retina and RPE-choroid homogenates from young (2-3 month old) albino rats were subjected to an iron-ascorbate-air pro-oxidant reaction medium, and compared to unoxidized control samples from young age-matched animals as well as senescent (24 month old) rats. In addition, neural retina and RPE-choroid homogenates from 3 month old albino rats were subjected to a 100% oxygen atmosphere to test whether the fluorescent products of autoxidation differ substantially from those generated in the pro-oxidant medium. The chloroform-soluble fluorophores of chloroform-methanol sample extracts were analyzed by corrected fluorescence spectroscopy and thin-layer chromatography (TLC). In vitro pro-oxidation of both the neural retina and the RPE from young rats produced blue-emitting fluorophores which differed from the orange- and yellow-emitting fluorophores extracted from the RPE of senescent rats. Corrected fluorescence spectroscopy of aged tissue extracts revealed vitamin A-related fluorescence (330 nm excitation maximum; 515 nm emission maximum) and a spectrally resolvable age-related fluorescence (420 nm excitation maximum; 600 nm emission maximum). Only the vitamin A-related fluorescence could be measured in the control of young samples.(ABSTRACT TRUNCATED AT 250 WORDS)

[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.

Retinoic binding in melanomas of the eye and in normal choroid.

Binding proteins for retinoic acid (cellular retinoid acid binding protein, CRABP), and for vitamin A (cellular retinol binding protein, CRBP) have been demonstrated in various cell types; these binding proteins display the characteristics of receptors. In the present study CRABP and CRBP levels were measured in 9 melanomas of the choroid. CRABP was detected in 2 of the melanomas, whereas CRBP was measurable in 1 melanoma. In comparison samples of normal choroid contained CRABP and CRBP in all cases investigated.

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.

Glycosaminoglycans in choroid.

Glycosaminoglycans (GAGs) purified from bovine choroidal tissues were identified and found to be composed of dermatan sulfate with smaller amounts of hyaluronic acid and heparan sulfate. Although biochemical analysis of unsaturated disaccharides (delta DiS) revealed the existence of delta Di-6S and delta Di-4S, corresponding GAG isomers were not identified as spots on the electrophoresis. Dermatan sulfate purified from the choroid seems to be a hybrid containing chondroitin sulfate C and chondroitin sulfate A.

13C solid-state cross polarization/magic-angle-spinning nuclear magnetic resonance spectra of natural and synthetic melanins.

The availability of NMR spectrometers operating in cross polarization/magic angle spinning (CP-MAS) has provided a powerful tool for the structural elucidation of insoluble materials. In this 13C NMR study of eumelanins we report the first direct evidence of the presence of different chemical functionalities in synthetic and natural eumelanins. These spectra contain useful information for the characterization of melanins from different sources.

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.

Melatonin-binding in the frog retina: autoradiographic and biochemical analysis.

Binding of melatonin was examined in the retina of Rana pipiens. When intact frog retinas were incubated with 3H-melatonin and processed for autoradiography, most of the radioactivity was localized to the melanosomes of the retinal pigment epithelium-choroid (RPE-choroid) and to the outer plexiform layer of the retina. Melanosome-enriched fractions of the RPE-choroid and membrane-enriched fractions of the neural retina demonstrated saturable melatonin binding when incubated with increasing melatonin concentration. Thin-layer chromatography showed that greater than 98% of the bound radioactivity was authentic melatonin. Scatchard analysis revealed a single population of binding sites with apparent Kd values of 6 X 10(-7) M for both the RPE-choroid and neural retina. When various indole analogs were tested for their ability to inhibit 3H-melatonin binding to the neural retina, both 5-methoxytryptophol and 6-chloromelatonin demonstrated complete displacement of melatonin binding. Endogenous retinal melatonin levels were measured by radioimmunoassay. A twofold increase in melatonin levels was observed during the dark period with peak levels at 384.5 +/- 28.8 pgms melatonin/pair retinas. Melatonin levels persisted in constant darkness, but were suppressed in constant light. Our data suggest that in the frog, the sites of action of retinal melatonin are the melanosomes of the RPE-choroid and the outer plexiform layer of the neural retina.

Retinal pigment epithelial lipofuscin and melanin and choroidal melanin in human eyes.

Optical measurements of the pigments of the retinal pigment epithelium (RPE) and choroid were made on 38 human autopsy eyes of both blacks and whites, varying in age between 2 wk and 90 yr old. Lipofuscin in melanin-bleached RPE was measured as fluorescence at 470 mm following excitation at 365 nm and was found to be proportional to fluorescence measured at 560 nm in unbleached tissue. Transmission measurements of RPE and choroidal melanin were converted and expressed as optical density units. The choroidal melanin content increased from the periphery to the posterior pole. RPE melanin concentration decreased from the periphery to the posterior pole with an increase in the macula. Conversely, the amount of RPE lipofuscin increased from the periphery to the posterior pole with a consistent dip at the fovea. There was an inverse relationship between RPE lipofuscin concentration and RPE melanin concentration. The RPE melanin content was similar between whites and blacks. Lipofuscin concentration was significantly greater (P = 0.002) in the RPE of whites compared to blacks; whereas blacks had a significantly greater (P = 0.005) choroidal melanin content than whites. The amounts of both choroidal and RPE melanin showed a trend of decreasing content with aging, whereas the amount RPE lipofuscin tended to increase (whites greater than blacks). Per fundus area, the amount of choroidal melanin was always greater than that in the RPE. There was a statistically significant (P = 0.001) increase in RPE height with age, most marked in eyes of whites after age 50 and correlated with the increase in lipofuscin concentration.

Lipid composition in melanosomes of tumor- and chorioidea cells.

The lipid composition of melanosomes from hamster and bovine eye pigmented tissues (chorioidea) and of melanosomes from Harding-Passey mouse melanomas, hamster melanomas Ma and human liver melanoma metastases was investigated. The lipid composition of melanosomes was analogous to that of other subcellular melanocytic fractions as it had been reported in literature. The main difference between tumor and chorioidea melanosomes consisted in the absence of phospholipids in the latter case, other differences were quantitative. The role of melanosomes in melanomas should not be different from that in normal pigmented tissues.

Feline maternal taurine deficiency: effects on retina and tapetum of the offspring.

The retina and tapetum of kittens born to taurine-deficient and taurine-supplemented mothers were compared. Retinal taurine concentrations typically reach adult levels 6 weeks postnatally. When measured at weaning at 8 postnatal weeks, the taurine concentrations in retina and tapetum of taurine-deficient kittens were 40% of normal levels. An ultrastructural correlate found in the retinas of taurine-deficient kittens was the presence of photoreceptor outer segments that were reduced in length and altered from the typical columnar configuration. Tapetal cells of taurine-deficient kittens were distinguished by accumulations of electron-dense droplets, the presence of tapetal rods with dilated limiting membranes and the presence of amorphous vesicles.

Experimental granulomatous uveitis: an electron microscopic study of pigment containing giant cells.

An experimental granulomatous uveitis in the Brown Norway rat is characterized by large numbers of giant cells and epithelioid cells containing uveal pigment. Ultrastructurally, the epithelioid cells and the giant cells exhibited melanosomes and individual melanin granules in the absence of phagocytic membranes and compound pigment granules. These observations support the view that the pigment containing giant cells may develop from uveal melanocytes.

Appearance of the noradrenergic markers tyrosine hydroxylase and neuropeptide Y in cholinergic nerves of the iris following sympathectomy.

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.

Algorithm for the correction of artifacts.

The sources of vitreous fluorophotometry artifacts are described. An algorithm to define this "spread function" is presented. The use of improved instrumentation and compensation for this artifact by data processing of the "correction" algorithm can improve the results of vitreous fluorophotometry. This algorithm was carried out in 40 eyes and was found to have an error of 0.7 mg/ml at 3 mm from the choroid-retina.