Synthesis and secretion of transferrin by a bovine trabecular meshwork cell line

The trabecular meshwork (TM) is the main outflow pathway in the mammalian eye. Oxidative damage to TM cells has been suggested to be an important cause of impairment of TM functions, leading to deficient drainage of aqueous humor, with deleterious consequences to the eye. Transferrin, a metalloprotein involved in iron transport, has been characterized as an intrinsic eye protein. Since transferrin is implicated in the control of oxidative stress, the objective of the present study was to determine if a bovine TM cell line (CTOB) synthesizes and secretes transferrin. The CTOB cell line was cultured in the presence of 35S-methionine and the incubation medium was submitted to immunoprecipitation. Total RNAs from CTOB and isolated bovine TM (freshly isolated, incubated or not) were subjected to the reverse transcription-polymerase chain reaction and the amplification products were sequenced. Also, both CTOB and histological TM preparations were processed for transferrin immunolocalization. A labeled peptide of about 80 kDa, the expected size for transferrin, was immunopurified from CTOB samples obtained from the incubation assays. The reverse transcription-polymerase chain reaction and sequencing experiments detected the presence of transferrin mRNA in CTOB and isolated bovine TM. Reactivity to antibodies against transferrin was observed both in CTOB and TM. The results obtained in all of these experiments indicated that the TM is capable of synthesizing and secreting transferrin. The possible implications for the physiology of the eye are discussed.

Synthesis and secretion of transferrin by a bovine trabecular meshwork cell line.

The trabecular meshwork (TM) is the main outflow pathway in the mammalian eye. Oxidative damage to TM cells has been suggested to be an important cause of impairment of TM functions, leading to deficient drainage of aqueous humor, with deleterious consequences to the eye. Transferrin, a metalloprotein involved in iron transport, has been characterized as an intrinsic eye protein. Since transferrin is implicated in the control of oxidative stress, the objective of the present study was to determine if a bovine TM cell line (CTOB) synthesizes and secretes transferrin. The CTOB cell line was cultured in the presence of 35S-methionine and the incubation medium was submitted to immunoprecipitation. Total RNAs from CTOB and isolated bovine TM (freshly isolated, incubated or not) were subjected to the reverse transcription-polymerase chain reaction and the amplification products were sequenced. Also, both CTOB and histological TM preparations were processed for transferrin immunolocalization. A labeled peptide of about 80 kDa, the expected size for transferrin, was immunopurified from CTOB samples obtained from the incubation assays. The reverse transcription-polymerase chain reaction and sequencing experiments detected the presence of transferrin mRNA in CTOB and isolated bovine TM. Reactivity to antibodies against transferrin was observed both in CTOB and TM. The results obtained in all of these experiments indicated that the TM is capable of synthesizing and secreting transferrin. The possible implications for the physiology of the eye are discussed.

A direct contact between astrocyte and vitreous body is possible in the rabbit eye due to discontinuities in the basement membrane of the retinal inner limiting membrane.

Different from most mammalian species, the optic nerve of the rabbit eye is initially formed inside the retina where myelination of the axons of the ganglion cells starts and vascularization occurs. Astrocytes are confined to these regions. The aforementioned nerve fibers known as medullated nerve fibers form two bundles that may be identified with the naked eye. The blood vessels run on the inner surface of these nerve fiber bundles (epivascularization) and, accordingly, the accompanying astrocytes lie mostly facing the vitreous body from which they are separated only by the inner limiting membrane of the retina. The arrangement of the astrocytes around blood vessels leads to the formation of structures known as glial tufts. Fragments (N = 3) or whole pieces (N = 3) of the medullated nerve fiber region of three-month-old male rabbits (Orictolagus cuniculus) were fixed in glutaraldehyde followed by osmium tetroxide, and their thin sections were examined with a transmission electron microscope. Randomly located discontinuities (up to a few micrometers long) of the basement membrane of the inner limiting membrane of the retina were observed in the glial tufts. As a consequence, a direct contact between the astrocyte plasma membrane and vitreous elements was demonstrated, making possible functional interactions such as macromolecular exchanges between this glial cell type and the components of the vitreous body.

A direct contact between astrocyte and vitreous body is possible in the rabbit eye due to discontinuities in the basement membrane of the retinal inner limiting membrane

Different from most mammalian species, the optic nerve of the rabbit eye is initially formed inside the retina where myelination of the axons of the ganglion cells starts and vascularization occurs. Astrocytes are confined to these regions. The aforementioned nerve fibers known as medullated nerve fibers form two bundles that may be identified with the naked eye. The blood vessels run on the inner surface of these nerve fiber bundles (epivascularization) and, accordingly, the accompanying astrocytes lie mostly facing the vitreous body from which they are separated only by the inner limiting membrane of the retina. The arrangement of the astrocytes around blood vessels leads to the formation of structures known as glial tufts. Fragments (N = 3) or whole pieces (N = 3) of the medullated nerve fiber region of three-month-old male rabbits (Orictolagus cuniculus) were fixed in glutaraldehyde followed by osmium tetroxide, and their thin sections were examined with a transmission electron microscope. Randomly located discontinuities (up to a few micrometers long) of the basement membrane of the inner limiting membrane of the retina were observed in the glial tufts. As a consequence, a direct contact between the astrocyte plasma membrane and vitreous elements was demonstrated, making possible functional interactions such as macromolecular exchanges between this glial cell type and the components of the vitreous body

A practical method for the radioisotope labeling of rabbit astrocytes with (3)H-thymidine.

A practical method for the radioisotope labeling aimed at the study of the proliferative behavior of astrocytes was described. It consisted in injecting 20 microCi of (3)H-thymidine into the vitreous body and tracing by autoradiography labeled astrocytes located both inside and outside the retina, e.g. optic nerve and neighboring parts of the central nervous system. The paraffin sections were immunostained for glial fibrillary acidic protein (GFAP) previous to autoradiographic processing. The semiquantitative analysis of labeled astrocytes was carried out on autoradiographs of semithin sections of rabbits killed as early as 6 h and as late as 3 months after the single intravitreal injection of (3)H-thymidine. Compared with the technique of labeling astrocytes by systemic administration (single injection or continuous infusion) of (3)H-thymidine into small animals, the method described herein has the following outstanding features: (i) it is much more economical in terms of the amount of labeled precursor used per animal; (ii) the labeling of the astrocytes is obtained as early as 6 h and remains up to 3 months after injection; (iii) the immunolabeling of the astrocytes is compatible with autoradiography; (iv) it is less risky to the experimental animal and to the environment; (v) it can be used in animals much larger than rats or mice.

An extensive system of extravascular smooth muscle cells exists in the choroid of the rabbit eye.

We investigated the structural organization of the choroid especially with regard to the presence of extravascular smooth muscle (EVSM) cells in albino rabbits. The eyes were fixed by intracardiac perfusion and processed for light, confocal, and electron microscopy. An unlabeled monoclonal antibody against alpha-actin of smooth muscle and a horseradish-peroxidase-conjugated secondary antibody were used for immunodetection of smooth muscle actin by light microscopy. For confocal microscopy, whole mount choroids were immunostained with a fluorescein isothiocyanate-conjugated (FITC) antibody. Our investigations revealed that the choroidal vessels are enveloped by bundles of EVSM. In contrast to the circular orientation of the smooth muscle cells of the tunica media of the choroidal vessels, the cells of the EVSM system were oriented longitudinally along the external surface of the vessel wall. The EVSM cells were strongly immunopositive for smooth muscle alpha-actin and exhibited a green fluorescence of the FITC-labeled anti-alpha-actin antibody. Individual cells were elongated and spindle-shaped, had the usual ultrastructural features of smooth muscle cells and, in places, were organized as 20 layers. EVSM cells were present throughout the thickness of the choroid, but not between the fenestrated endothelial lining of the choriocapillaris and Bruch's membrane, and extended from the optic nerve to the ciliary body where they merged with the ciliary muscles. Based on the three dimensional organization, immunoreactivity, and cellular and subcellular features of the EVSM system as well as information in the literature, we hypothesize that, functionally, this system, in conjunction with the choroidal vasculature, contributes to the myogenic control of choroidal blood flow and tissue volume, and also affects the intraocular pressure as well as the refractive and accommodative state of the eye.

[Application of confocal microscopy in the study of the relationships between astrocytes and blood vessels in the retina]. / Aplicación de la microscopia confocal al estudio de las relaciones vaso-gliales en la retina.

OBJECTIVE: To study the rabbit retina astrocytes by using immunofluorescence techniques together with the confocal microscopy. MATERIAL AND METHODS: The rabbit retinas were processed with an anti-glial fibrillary acidic protein (anti-GFAP) for astrocyte detection and with propidium lobide for nuclear staining. RESULTS: Confocal microscopy allows for three-dimensional reconstruction of astroglial cells, the performance of double staining procedures with superposition of images corresponding to each chromogen, the exchange of observation axes for each slide and finally the performance of serial optic sections that indicate the exact cell location and their relationship with adjacent structures, eliminating the background signals. CONCLUSIONS: The confocal microscopy provides detailed information about tridimensional morphology and the location of the astrocytes in the rabbit retina. The astrocytes associated with the nerve fiber bundles are located in the nerve fiber layer. The type III perivascular astrocytes are located between the intravitreous capillaries close to the internal limiting membrane of the retina. The type I perivascular astrocytes are found in the retinal face of the intravitreal capillaries, these being the neurons which are the most distant from the retina.

Aplicación de la microscopia confocal al estudio de las relaciones vaso-gliales en la retina / Application of confocal microscopy in the study of the relationships between astrocytes and blood vessels in the retina

Objetivo: Se pretende estudiar los astrocitos de la retina del conejo utilizando técnicas inmunohistoquímicas de fluorescencia asociadas a la microscopia confocal. Material y métodos: Se analizan mediante microscopia confocal retinas de conejo teñidas con anti-GFAP. para el marcaje de los astrocitos, y con ioduro de propidio para marcaje de los núcleos Resultados: El empleo del microscopio confocal permite la reconstrucción tridimensional de las células astrogliales, la realización de dobles marcajes superponiendo las imágenes obtenidas con cada uno de ellos, el cambio de los ejes de observación y toma de imágenes de la preparación, y por último la realización de series de secciones ópticas que nos indican la localización exacta de las células y sus relaciones con las estructuras adyacentes, eliminando las señales de fondo. Conclusión: El estudio de la retina del conejo con microscopia confocal proporciona una información detallada de la morfología tridimensional y de la localización de los astrocitos. Los astrocitos acompañantes de axones se localizan en el espesor de la capa de fibras del nervio óptico. Los astrocitos perivasculares tipo III se sitúan en el humor vítreo en la proximidad de la membrana limitante interna de la retina. Los astrocitos perivasculares tipo I se encuentran en la cara retiniana de los vasos intravítreos siendo la célula nerviosa más alejada de la retina (AU)

Immunohistochemistry in association with scanning electron microscopy for the morphological characterization and location of astrocytes of the rabbit retina.

The purpose of the present investigation was to establish a method for the morphological characterization and location of the several types of astrocytes in the rabbit retina. Whole retinas were incubated with unlabeled antibody to glial fibrillary acidic protein (GFAP) and, afterwards, treated with secondary antibody labeled according to the requirements for the visualization of the antigen-antibody reaction either with the confocal or transmission electron microscope. Specimens treated similarly to the latter were osmium enhanced and analyzed with scanning electron microscopy (SEM). The different immunohistochemical approaches led to the conclusion that the cells selectively visualized with the SEM are astrocytes. The higher resolution and depth of focus of this instrument allowed a better morphological characterization and a more precise location of the astrocytes in the several levels of the inner portion of the rabbit retina. The method described herein, in which pre-embedding immunohistochemistry for GFAP on rabbit retinas was associated with osmium enhancement and examination with SEM, proved to be reliable and efficient for the morphological characterization and location of astrocytes.

Glycosaminoglycans in components of the rabbit eye: synthesis and characterization.

PURPOSE: To trace the eye components involved in proteoglycan synthesis and to characterize the sulfated glycosaminoglycans which are associated to these macromolecules. METHODS: Sodium [(35)S]-sulfate was injected intravitreally and the rabbits were killed at different time intervals after the injection. The glycosaminoglycans of choroid, ciliary body, cornea, iris, lens capsule, retina and sclera were extracted and processed for estimations of their specific activities, and for electrophoresis plus autoradiography with or without previous treatment with specific enzymes. In addition, methacrylate sections of the eyes were analysed by autoradiography. RESULTS: The peak of specific activities of the glycosaminoglycans of all eye components occurred at 2 days after the intravitreal injection of [( 35)S]-sulfate. The autoradiography of the agarose gels revealed three types of glycosaminoglycans, namely, heparan-, chondroitin- and dermatan sulfate, only in the retina. The other eye components contained heparan sulfate and either chondroitin or dermatan sulfate. Tissue autoradiography together with the biochemical techniques contributed to unravel the origin of the glycosaminoglycans in the eye components. CONCLUSIONS: The results of the present investigation have shown that heparan sulfate, contrasting to chondroitin sulfate and dermatan sulfate, is synthesized in all eye components studied and that the glycosaminoglycan composition differs according to the tissue of origin.

Sulfation of intrinsic glycoproteins of the rabbit vitreous.

The experiments reported here were designed to characterize the intrinsic vitreous glycoproteins and to understand the process of their sulfation. Rabbits were injected intravitreally with 35S-sodium sulfate and killed at several time intervals after injection. In another series of experiments, rabbits were injected either with 35S-sodium sulfate, 3H-fucose or 3H-tyrosine, associated or not associated with tunicamycin administration. Vitreous from the control eyes was also digested with N-glycosidase. Furthermore, ciliary bodies, the putative source of the intrinsic vitreous glycoproteins, were incubated with 35S-sodium sulfate in the presence or absence of the protein synthesis inhibitor cycloheximide, and the culture media recovered for analysis. These and the vitreous samples of the other experiments were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Except for serum albumin, practically all polypeptide bands of the vitreous and culture media were labeled with radioactive sulfate and were shown to undergo renewal. The experiments using tunicamycin or enzyme treatment suggest that radioactive sulfate was incorporated not only into the carbohydrate side chains of the glycoproteins but also into the amino acid tyrosine of the polypeptide backbone of these glycoproteins.

Transferrin production by the ciliary body of rabbits: a biochemical and immunocytochemical study.

PURPOSE: We have previously reported that transferrin is one of several glycoproteins synthesized within the eye and secreted into the vitreous. The present investigation was designed to determine the role of the ciliary body in the production of this vitreous transferrin. METHODS: Isolated ciliary body-iris were incubated with 3H-fucose, 3H-tyrosine or 35S-methionine and afterwards the culture media were processed for affinity chromatography using columns of Sepharose conjugated with antibody to rabbit plasma transferrin. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using total RNA extracted from fresh ciliary body-iris and primers constructed on the basis of the known sequence of transferrin mRNA from rabbit liver. The fragment obtained was employed as a probe in northern-blots of total RNA of ciliary body-iris. Furthermore, paraffin sections of eyes were treated for immunocytochemical visualization of transferrin. RESULTS: A labeled polypeptide, specifically eluted from the antitransferrin columns, was detected in the incubation medium, transferrin mRNA was found in extracts of whole ciliary body-iris, and transferrin antigenicity was identified in the ciliary and iridial epithelial cells by immunocytochemistry. CONCLUSIONS: These results demonstrate the ciliary epithelium as one of the sources of the vitreous transferrin.

Biochemical studies on the secretion of glycoproteins by isolated ciliary body of rabbits.

The contribution of the ciliary body to the origin of vitreous proteins was investigated in rabbits by incubating explants of this eye component under novel conditions. At the end of incubations for up to 21 h, the tissues were processed histologically and were shown to be in an excellent state of morphological preservation. When radioactive amino acids and fucose were added to the culture medium, protein and glycoprotein synthesis and secretion were detected using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) plus fluorography. The origin of these secretory products was traced by autoradiography to the ciliary epithelium. When samples of vitreous bodies - injected intravitreally with the same radioactive precursors - were run beside samples of the tissue culture media, comigration of at least 8 radioactively labelled bands including the one previously identified as transferrin was detected. This indicates that some vitreous proteins may be secreted by the ciliary body and that cultures of explants of ciliary body-iris are useful tools for studies on vitreous protein secretion.

Transferrin, one of the major vitreous proteins, is produced within the eye.

Transferrin occurs in the vitreous at a higher relative concentration than found in the plasma or in the aqueous humor. This has been related to a possible local synthesis of transferrin by some component of the eye, although convincing evidence for this has not been available. Recently, the intraocular synthesis of several vitreous glycoproteins, possibly by the ciliary body, was demonstrated by our group. This paper reports experiments that characterize vitreous transferrin as one of these glycoproteins. Vitreous of rabbits injected intravitreally either with 3H-fucose or 3H-tyrosine and killed 40 hr after the injection was processed for 1D and 2D-electrophoresis, followed by immunoblot techniques or fluorography. It was possible to detect a labeled polypeptide with the same molecular weight and pI of rabbit plasma transferrin. Furthermore, this labeled polypeptide could be specifically eluted from columns of Sepharose conjugated with antibody against rabbit plasma transferrin. Thus, these results demonstrate that at least part of the transferrin of the vitreous is synthesized within the eye.

Studies on the origin of the glycoproteins of the rabbit vitreous body using a protein-synthesis inhibitor and radioactive fucose and amino acids.

Rabbits were injected intravitreally with [3H]-fucose and were killed at 0.5, 4, 24, and 40 h postinjection. The radiometric measurements and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) plus fluorography performed on the vitreous bodies of these rabbits were compared with those carried out on excised vitreous bodies of noninjected rabbits incubated with [3H]-fucose for the same periods. The results of these in vitro experiments demonstrated that at all test intervals there were only two lightly labeled bands in the fluorograms, and this was interpreted as nonenzymatic binding of [3H]-fucose to proteins (glycation), occurring in the extracellular environment. In contrast, intravitreal injections resulted in about 14 conspicuously labeled bands, mainly after the longer time intervals, and the nature of these bands was revealed by additional experiments. In another series of experiments, rabbits were injected intravitreally with either [3H]-fucose or [3H]-amino acids and their vitreous bodies were processed as described above. The specific activity (counts per minute per microgram of protein) of the glycoproteins labeled with [3H]-fucose was always higher than that of the proteins labeled with any of the amino acids. In fluorograms, eight bands unequivocally labeled with the [3H]-amino acids were detected, and corresponding bands almost invariably appeared in the [3H]-fucose lane. Cycloheximide, a protein-synthesis inhibitor, blocked almost completely the incorporation of both [3H]-proline and [3H]-fucose into the vitreous proteins as revealed by the radiometric measurements and fluorography.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiographic, electrophoretic, and immunocytochemical studies of glycoproteins of the rabbit iris.

L-[3H]fucose was injected either intravitreally or intra-aqueously into adult rabbits which were killed at several time points after injection. SDS-polyacrylamide gel electrophoresis and fluorography of iris extracts revealed that most of the proteins are glycoproteins containing fucose residues. Autoradiography of semi-thin histologic sections demonstrated that glycoprotein synthesis was most prominent in the epithelium of the iris, while little protein synthesis was evident in the stroma of the iris. The results of these experiments indicated that the glycoproteins of the iris undergo renewal. The protein band pattern of the iris extracts was very similar to that of extracts of the ciliary body. The high-molecular-weight cartilage matrix glycoprotein (CMGP), an intrinsic component of the ciliary body, vitreous, and aqueous humor, was detected by immunohistologic studies only in the stroma of the iris. The results of immunohistochemical analyses of the eyes of young rabbits (1-21 days old), in addition to the autoradiographic findings, strongly suggest that CMGP is not an intrinsic glycoprotein of the iris stroma, at least in this species.

Origin and renewal of the intrinsic glycoproteins of the aqueous humor.

Rabbits were injected either intravitreally or intra-aqueously with L-[3H]-fucose and killed at several intervals after the administration of this marker for glycoproteins. The aqueous humor, the vitreous body and the ciliary body were processed for radiometry (liquid scintillation counting), sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and fluorography. Light microscopic autoradiography was carried out on semi-thin sections of the ciliary body and revealed intense activity in terms of the synthesis, migration and renewal of glycoproteins in the ciliary epithelium. The amount of unbound [3H]-fucose in the aqueous humor decreased sharply by 4 h, and the labeled glycoproteins were present only in very small quantities at 1 day after the intra-aqueous injection. When [3H]-fucose was injected intravitreally, unbound radiolabel could be detected in the aqueous humor for greater than 1 day and the labeled glycoproteins, for up to 21 days after injection. The amount of unbound or bound [3H]-fucose was higher in the vitreous than in the aqueous at any interval after the intravitreal injection. Following the intra-aqueous injection, the amount of label that reached the vitreous body was practically insignificant. The levels of radioactivity in the serum were extremely low, as they were in the contralateral eye when tritiated fucose was injected into one eye only. Most of the Coomassie blue-stained bands detected in SDS-PAGE contained labeled glycoproteins as revealed by fluorography of gels simultaneously containing aqueous and vitreous samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid phosphatase activity in mature secretory granules of the salivary gland of Bradysia hygida.

It is uncommon to find acid phosphatase activity in mature secretory granules. This paper demonstrates by light and electron microscope cytochemistry an acid phosphatase in mature secretory granules in the cells of one region of the salivary gland of Bradysia hygida (Diptera, Sciaridae). These secretory granules increase in number during larval development up to the beginning of the pre-pupal period when they undergo massive exocytosis. Biochemical assays show that upon exocytosis of the majority of the granules the total acid phosphatase activity in the granular gland region drops to 10% of the maximum reached before exocytosis. During and after exocytosis, two other acid phosphatases, eletrophoretically different and much weaker in activity, become increasingly detectable in all gland regions. At the same time, in whole mount preparations, numerous tiny acid phosphatase-positive granules (probably secondary lysosomes) become evident in all major cell types of the salivary gland. These results indicate that the S2 region of the salivary gland has mature secretory granules containing an acid phosphatase destined for exocytosis which is different in molecular properties from other acid phosphatases (likely lysosomal) made by the gland.

Partial characterization, origin and turnover of glycoproteins of the rabbit vitreous body.

L-[3H]fucose was injected intravitreally into rabbits that were killed from 1 hr to 28 days after injection. The vitreous bodies were processed for radiometric techniques, and electrophoresis followed by fluorography. Unbound [3H]fucose remained at a high level up to 1 day, whereas the peak of [3H]fucose bound to glycoproteins was observed at 3 days after injection with a continuous decrease afterwards. The turnover rate of vitreous glycoproteins was estimated at 4.37% per day and their turnover time at 22.85 days. Electrophoresis and fluorography combined revealed about 14 bands of glycoproteins with fucose residues and there were strong indications of differences in turnover rate among individual glycoproteins. The most prominent band in the Coomassie blue-stained gels was the one having a molecular weight of 69 kDa and it was not labeled with [3H]fucose. This band was tentatively identified as serum albumin. On the other hand, the 14 bands labeled with [3H]fucose were glycoproteins originating from within the eye, that is, they are intrinsic constituents of the vitreous body.

The origin of the intrinsic glycoproteins of the rabbit vitreous body: an immunohistochemical and autoradiographic study.

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.