Heterogeneity of the ciliary epithelium of the rat eye as revealed by spot 35 protein (a Purkinje cell specific protein)-like immunoreactivity

By means of immunoreactivity for spot 35 protein, a novel cerebellar Purkinje cell-specific protein, the regional heterogeneity among non-pigmented ciliary epithelial cells of rats was demonstrated with reference to the antero-posterior and crest-valley directions of individual ciliary epithelial folds in immature and mature eyes. The functional significance of the occurrence of the spot 35 immunoreactivity in the posterior portion of the ciliary epithelium is briefly discussed in relation to the formation of the aqueous humor.     

Heterogeneity of the ciliary epithelium of the rat eye as revealed by spot 35 protein (a Purkinje cell specific protein)-like immunoreactivity

Summary By means of immunoreactivity for spot 35 protein, a novel cerebellar Purkinje cell-specific protein, the regional heterogeneity among non-pigmented ciliary epithelial cells of rats was demonstrated with reference to the antero-posterior and crest-valley directions of individual ciliary epithelial folds in immature and mature eyes. The functional significance of the occurrence of the spot 35 immunoreactivity in the posterior portion of the ciliary epithelium is briefly discussed in relation to the formation of the aqueous humor.     

Internalized gap junctions in ciliary epithelium of rabbit and rat

Summary Transmission electron microscopy was used to examine internalized gap junctions (IGJ) in rabbit and rat ciliary epithelial cells. A prominent feature of all the specimens studied was the presence of different images of IGJ membrane that entrapped a portion of an adjoining cell. We documented and analyzed more than 500 gap junction (GJ) vacuoles and invaginations, the latter comprising less than 20% of all the structures examined. With ten exceptions found in non-pigmented cells, all the IGJ were unidirectionally internalized within the cytoplasm of pigmented epithelial cells. Morphological signs of autophagic degradation of GJ vacuoles were observed. An essential finding was that once a GJ membrane started to invaginate, a lucidation of a part of the protruding cytoplasm occurred; no planar GJ membranes exhibited such an alteration. The present findings suggest that IGJ derived from the epithelium of ciliary processes arise through an invagination-endocytosis mechanism and are degraded autophagically. This phenomenon may be relevant to aqueous humor production.     

The endothelial pocket

Summary A new endothelial cell structure, named the endothelial pocket, has been found by combined transmission and scanning electron microscopic studies of renal peritubular capillaries. Transmission EM observations made on these and other fenestrated capillaries demonstrated that each pocket consists of an attenuated fold of fenestrated endothelium that projects 200 nm into the lumen above the rest of the endothelial surface. Beneath this luminal fold, there is a space and then another layer of fenestrated endothelium which abuts the basal lamina. The linear density of endothelial pockets was measured in the capillaries of the kidney cortex, intestinal mucosa and exocrine pancreas in mice and determined to be 0.067, 0.017 and 0.007 pockets·m^-1 respectively. Cationic ferritin decoration of the anionic sites on the luminal surface of the endothelium in these capillary beds revealed that both unlabelled and labelled diaphragms are clustered. In such specimens, the majority of the luminal diaphragms on endothelial pockets did not have cationic ferritin binding sites detectable by either scanning or transmission EM. On this account as well as on account of their general morphology, endothelial pockets appear to be multifold versions of the simple transendothelial channel.     

Charged sieving by the basal lamina and the distribution of anionic sites on the external surfaces of fat body cells

The distribution of anionic sites on the basal lamina has been examined with highly cationic ferritin. The penetration of ferritins, with a range of charges from anionic to highly cationic, through the basal lamina into the spaces between fat body cells in an insect is correlated with the charge of the tracer. The anionic sites of the basal lamina may therefore affect the composition of the lymph that bathes the fat body cells. There was more cationic ferritin bound to the plasma membrane reticular reticular system than to the lateral plasma membranes, suggesting that there may be regional differences in surface charge.     

High glucose-6-phosphatase activity in non-pigmented epithelial cells of rabbit ciliary body.

For study of the origin of glucose in the aqueous humor, glucose-6-phosphatase (G6Pase) and hexokinase activities, and glycogen, were cytochemically examined in the ciliary body (CB) of rabbit. G6Pase activity was also assayed biochemically. The staining reaction for G6Pase activity was strong in the non-pigmented epithelium (NPE) in the pars plana and tips of ciliary processes in the region containing large ciliary pockets within the pars plicata. NPE cells contained abundant reaction product for G6Pase activity in the endoplasmic reticulum (ER) and nuclear envelope. However, NPE in other regions of the CB and pigmented epithelium (PE) of CB, and other areas surrounding the anterior and (PE) of CB, and other areas surrounding the anterior and posterior chambers, showed weak or no G6Pase staining reaction. Biochemical G6Pase activity in the whole ciliary body was relatively high. Both NPE and PE in the pars plana and the tips showed strong staining reaction for hexokinase activity but no staining for glycogen. Furthermore, NPE cells in the tips bore large aggregates of smooth ER and many Golgi apparati. These suggest that the high G6Pase activity in NPE cells in the pars plana and the tips is related to glucose release into the aqueous humor.     

Effect of molecular charge on choroid-plexus permeability: Tracer studies with cationized ferritins

Summary The permeability of the choroid plexus and renal glomerulus to intravenously injected native, anionic ferritin and various cationic ferritin derivatives was studied in normal rats by electron microscopy. In both structures, anionic, native ferritin was largely confined to the circulatory compartment while the cationic forms penetrated and accumulated within the filtration barriers. In the choroid plexus, cationic ferritin concentrated in relationship to the endothelial fenestrations and the subendothelial basal lamina region. In the glomerulus, there was also an inner concentration of cationic tracer and, in addition, an aggregation of tracer along the outer, subepithelial portion of the basal lamina. The results indicate that the localization of tracer within the filtration barrier of both the choroid plexus and renal glomerulus is directly related to the tracer's isoelectric point. The findings suggest that the choroid plexus, like the glomerulus, contains fixed anionic groups within the capillary wall which influence its permeability.     

Localization and ontogeny of aquaporin-1 and -4 expression in iris and ciliary epithelial cells in rats

The precise localization of aquaporin (AQP)1 and AQP4 was studied in iris and ciliary epithelial cells, in both mature and developing rats, to elucidate the molecular mechanisms underlying aqueous humor balance. Anterior segments of eyes dissected from embryonic day (E)13, E15, E18, and E20, postnatal day (P)0, P7, and P14, and postnatal week 8 rats were subjected to immunofluorescence analysis with AQP isoform-specific antibodies. In adult rat eye, AQP1 was localized to the apical and basolateral plasma membranes of iris epithelial cell layers and of anterior ciliary non-pigmented epithelial (NPE) cells. Conversely, AQP4 was localized to the basolateral plasma membrane of NPE cells in ciliary epithelium and the posterior iris. Developmentally, AQP1 was detected as early as E15 in immature iris and ciliary epithelial cells, and expression persisted throughout development up to adulthood. In contrast, AQP4 was first observed at P7 in the developing pars plicata, and the AQP4-positive area gradually spread to cover the entire pars plicata as development proceeded. These findings indicate that both AQP1 and AQP4 contribute to aqueous humor secretion in the rat eye, thereby maintaining proper intraocular pressure. Moreover, AQP appears to play a major role in aqueous humor secretion in early eye development. This study thus provides a basis for understanding the molecular mechanisms of aqueous humor secretion in pathological and physiological conditions.     

Basis of Chloride Transport in Ciliary Epithelium

The aqueous humor is formed by the bilayered ciliary epithelium. The pigmented ciliary epithelium (PE) faces the stroma and the nonpigmented ciliary epithelium (NPE) contacts the aqueous humor. Cl⁻ secretion likely limits the rate of aqueous humor formation. Many transport components underlying Cl⁻ secretion are known. Cl⁻ is taken up from the stroma into PE cells by electroneutral transporters, diffuses to the NPE cells through gap junctions and is released largely through Cl⁻ channels. Recent work suggests that significant Cl⁻ recycling occurs at both surfaces of the ciliary epithelium, providing the basis for modulation of net secretion. The PE-NPE cell couplet likely forms the fundamental unit of secretion; gap junctions within the PE and NPE cell layers are inadequate to maintain constancy of ionic composition throughout the epithelium under certain conditions. Although many hormones, drugs and signaling cascades are known to have effects, a persuasive model of the regulation of aqueous humor formation has not yet been developed. cAMP likely plays a central role, potentially both enhancing and reducing secretion by actions at both surfaces of the ciliary epithelium. Among other hormone receptors, A₃ adenosine receptors likely alter intraocular pressure by regulating NPE-cell Cl⁻ channel activity. Recently, functional evidence for the regional variation in ciliary epithelial secretion has been demonstrated; the physiologic and pathophysiologic implications of this regional variation remain to be addressed.This revised version was published online in June 2005 with a corrected cover date.     

Cationic ferritin binding sites and surface charge densities of transformed cells

Bioelectric surface properties of the high and low tumor-producing cell lines, NCTC 2472 and NCTC 2555, respectively, were determined by cationic ferritin binding and the electrophoretic mobility of intact cells. Measurements of anionic sites were bases on the number of cationic ferritin particles per 0.01 mu 2 that were electronically tagged and counted by an image analyzer. The average particle count was 45 for the control "high" cells and 34 for the control "low" cells. The surface charge densities, expressed as electrostatic units per cm-2 x 10(-13) were 2.34 and 1.18 at 50 V (2 mA) for the "high" and "low" control cells, respectively. Enzymic cleavage of sialic acid and other carbohydrate moieties resulted in up to an 81% reduction in the charge densities and a 57% reduction of the anionic sites of the "high" cells. The electrophoretic mobility of cells with bound cationic ferritin showed that up to 50% of the exposed anionic sites fail to bind cationic ferritin. Preliminary findings on the particle size/distribution by image analysis showed wide ranges in both particle size and interparticle distances that may limit cationic ferritin binding.     

Adenosine,adenosine receptors and glaucoma: An updated overview

Background

Glaucoma, a leading cause of blindness worldwide, is an optic neuropathy commonly associated with elevated intraocular pressure (IOP). The major goals of glaucoma treatments are to lower IOP and protect retinal ganglion cells. It has been revealed recently that adenosine and adenosine receptors (ARs) have important roles in IOP modulation and neuroprotection.

Scope of review

This article reviews recent studies on the important roles of adenosine and ARs in aqueous humor formation and outflow facility, IOP and retinal neuroprotection.

Major conclusions

Adenosine and several adenosine derivatives increase and/or decrease IOP via A_2A AR. Activation of A₁ AR can reduce outflow resistance and thereby lower IOP, A₃ receptor antagonists prevent adenosine-induced activation of Cl⁻ channels of the ciliary non-pigmented epithelial cells and thereby lower IOP. A₁ and A_2A agonists can reduce vascular resistance and increase retina and optic nerve head blood flow. A₁ agonist and A_2A antagonist can enhance the recovery of retinal function after ischemia attack. Adenosine acting at A₃ receptors can attenuate the rise in calcium and retinal ganglion cells death accompanying P2X(7) receptor activation.

General significance

Evidence suggested that the adenosine system is one of the potential target systems for therapeutic approaches in glaucoma.     

DARPP-32 in the ciliary epithelium of the eye: a neurotransmitter-regulated phosphoprotein of brain localizes to secretory cells

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.     

Ultracytochemistry of cholera-toxin binding sites in ciliary processes

Summary Cholera toxin reduces the rate of formation of aqueous humor in concentrations (10^–11 M) that do not disturb the morphology of the aqueoushumor forming epithelial cells of the ciliary processes of the rabbit eye. The search for an endogenous mediator of aqueous-humor formation comparable to cholera toxin in its mode of operation prompted us to map the distribution of cell surface receptors for cholera toxin in the ciliary processes of the eyes of rabbits. Cytochemical studies were carried out with the use of conjugates of cholera toxin to fluorescein isothiocyanate (CT-FITC) and to horseradish peroxidase (CT-HRP), and of the B subunit of cholera toxin to horseradish peroxidase (B-HRP). Multiple fluorescent CT-FITC binding sites were observed on the outer nonpigmented epithelial layer near the crests of the processes. Processes incubated with CT-HRP in vitro showed surface staining of 30–40% of the nonpigmented epithelial cells. A prominent reaction product was observed along the basal and lateral plasma membranes of these cells. In vivo studies carried out after arterial infusion of B-HRP showed a reproducible dense reaction product between the apical surfaces of the pigmented epithelium (PE) and of the nonpigmented epithelium (NPE) facing each other. Aggregations of reaction product were observed with the electron microscope in the extracellular space between the apices of PE and NPE. The apical plasma membrane of the endothelium of the blood vessels near the crests of the ciliary processes was stained after either in vivo or in vitro exposure to peroxidase conjugates. These findings indicate that the cell-surface receptors which mediate the action of cholera toxin on aqueous humor formation are very likely localized in the apical plasma membranes of the epithelium of the ciliary processes.Supported in part by USPHS grant # EY-00237, the Connecticut Lions Eye Research Foundation, Inc., and Research to Prevent Blindness, Inc.     

Connexin 43 and the glucose transporter,GLUT1, in the ciliary body of the rat

To investigate the relationship between the gap junction protein connexin 43 and the glucose transporter GLUT1, their localization was visualized by double-immunofluorescence microscopy using frozen sections as well as immunogold staining of ultrathin frozen sections. In pigmented epithelial cells, most of the GLUT1 was localized along the plasma membrane facing the blood vessels, whereas in non-pigmented epithelial cells. it was present along the plasma membrane facing the aqueous humor. Connexin 43 was abundant in the ciliary body and localized mainly in the gap junctions connecting the pigmented and non-pigmented epithelial cells. Localization of GLUT1 and connexin 43 in the blood-aqueous barrier suggests that GLUT1, connexin 43, and GLUT1 disposed in this order could be a machinery responsible for the transport of glucose across the blood-aqueous barrier.     

Effect of charge density of cationic polyelectrolytes on complex formation with DNA

The interaction between DNA and ionen polymers, -[N⁺(CH₃)₂(CH₂)_mN⁺(CH₃)₂(CH₂)_n], with m-n of 3–3, 6–6, and 6–10 were examined in order to know how the binding behavior of cationic polymers with DNA depends on the charge density of polycation. The ionen polymer has no bulky side chain and the binding forces with DNA would be attributed mainly to electrostatic interaction. When 3–3 ionen polymers were added to DNA solution, precipitable complexes with the ratio of cationic residue to DNA phosphate (+/?) of 1/1 and the free DNA molecules were segregated, while 6–6 and 6–10 ionen polymers formed soluble complexes with DNA molecules up to (+/?) = 0.5. This suggests that 3–3 ionen polymers bind cooperatively with DNA while 6–6 and 6–10 ionen polymers bind noncooperatively. The cooperative binding of 3–3 ionen polymer and the noncooperative binding of 6–6 ionen polymer were also supported by the thermal melting and recooling profiles from the midpoint between first and second meltings. It was concluded that the charge density of DNA phosphate is a critical value determining whether the ionen polymers bind to DNA by a cooperative or by a noncooperative binding, since the distance between successive cationic charges of 3–3 ionen polymer is shorter than that between successive phosphate charges on DNA double helix and those of 6–6 and 6–10 ionen polymers are longer.     

Distribution patterns in glycoconjugate expression during the development of the rat palate

Summary The distribution of complex carbohydrate structures during the embryonic development of the rat palate was analysed by examining lectin-binding patterns in serial paraffin and cryostat sections. With few exceptions, the binding patterns showed a general increase in lectin receptors in the more developed stages of palatogenesis. High mannose oligosaccharides were especially amplified during development. Terminal fucose molecules were not expressed. In contrast, terminal sialic acid molecules were ubiquitously distributed in epithelial and mesenchymal tissues. Non-sialylated terminal N-acetylglucosamine was specifically restricted to evolving bone matrix. Before palatal fusion, quantitative but not qualitative differences were detected between oral, nasal, and medial-edge epithelial surfaces. The only exception was LCA, which specifically marked epithelial cells at the tip of palatal shelves. A very selective affinity for Jacalin was demonstrated in the oral epithelium of the palate after day 16, suggesting the presence of sialylated terminal galactose-(-1,3)-N-acetylgalactosamine. PNA specifically marked the basal lamina of the oral side of palatal processes. The binding patterns of DBA, GSL I_A, SBA, and VVA indicated that the epithelium of the tongue is characterized by terminal - and -galactose residues, whereas palatine cells possess only molecules with -anomery. During palatogenesis, glycosaminoglycans patterns were significantly modified. Our data suggest that alteration of complex carbohydrate structures may play a central role in modulating cell-cell and cell-matrix interactions. The significance of these findings, however, remains to be elucidated.     

Orthogonal arrays of particles in non-pigmented cells of rat ciliary epithelium: Relation to distribution of filipin- and digitonin-induced alterations of the basolateral membrane

Summary It has been suggested that orthogonal arrays of particles may increase the rigidity of plasma membrane, as does cholesterol. Therefore, using freeze-fractured non- pigmented ciliary epithelium, the distribution of such arrays was compared to the distribution of membrane deformations induced by the sterol-probes filipin and digitonin in different domains of the basolateral plasma membrane. The distribution of orthogonal arrays of particles was homogeneous between different regions of the basolateral membrane of the non-pigmented ciliary epithelium, while the number of filipin-induced alterations was nearly 4 times higher in the membrane domains not in contact with the basal lamina than in domains in contact with it. Contrary to the homogeneous distribution of arrays, digitonin-induced deformations also differed markedly in these two basolateral membrane domains. Considering that a marked positive response to sterol probes implies a high sterol content, we conclude that orthogonal arrays of particles can occur in plasma membrane regions well-provided with cholesterol and not in direct contact with the basal lamina. Other possible roles of these arrays are discussed.This paper was presented in part at the ARVO meeting, April– May 1986, Sarasota, USA     

Uptake and intracellular transport of cationic ferritin in the bronchiolar and alveolar epithelia of the rat

Summary Cationic ferritin was used as a marker to reveal the processes of endocytosis and intracellular transport in bronchiolar and alveolar epithelia. The marker was injected into the lung via the trachea, and ultrastructural observation of the distribution of ferritin particles in bronchiolar and alveolar epithelial cells was carried out at intervals of 5, 15, 30 and 60 min after the injection. The luminal surface of the airway and the alveolar epithelium showed diffuse labeling with cationic ferritin. In general, ferritin particles were observed in vesicles and vacuoles of the bronchiolar and alveolar epithelial cells within 5 min of injection; they appeared in multivesicular bodies within 15 min. Multivesicular bodies and secondary lysosomes containing ferritin particles, some of which showed a positive reaction for acid phosphatase, were seen in the basal cytoplasm within 30 min; ferritin particles appeared in the basal lamina below the Clara cells, ciliated cells and type 2 alveolar cells within 30 min. Ferritin particles were seen in ovoid granules of some Clara cells and in lamellar inclusion bodies of many type 2 alveolar cells. Brush cells and type 1 alveolar cells took up only a small quantity of ferritin particles.     

The blood-brain barrier in a reptile, Anolis carolinensis

An electron microscopic study was made of the ultrastructure and permeability of the capillaries in the cerebral hemispheres of the lizard, Anolis carolinensis. The brain of Anolis is vascularized by a loop-type pattern consisting exclusively of arteriovenous capillary loops. The ultrastructure of the endothelium and the arrangement of the various layers from the capillary lumen to the central nervous tissue is similar to that of mammals. The endothelial cells form a continuous layer around the lumen and are joined by tight interendothelial junctions. The basal lamina of the endothelium is also continuous and encloses pericyte processes. The cells of the nervous tissue rest directly on the basal lamina of the capillary and are separated from each other by a 200 Å space. Intravenously injected horseradish peroxidase (MW 40,000) and ferritin (MW 500,000) were used to study the permeability of the capillaries. The entry of horseradish peroxidase and ferritin into the intercellular spaces of the brain is restricted by the tightness of the interendothelial junctions. No vesicular transport of either tracer occurs; however, ferritin does enter the endothelial cells in vacuoles. No tracer molecules are present in the basal lamina, pericytes, or nervous tissue. The different responses of the endothelial cell to the tracers used in this study suggest that endocytotic activities of endothelial cells involve different processes. Vacuoles formed by marginal folds, vacuoles formed by endothelial surface projections or deep invaginations of the plasma membrane, 600–800 Å vesicles, and coated vesicles all seem to differ in the nature of the substances which they endocytose.     

Effects of gamma irradiation on BCL2 and TPR53BP2 expression in the porcine ciliary body.

BACKGROUND: When dissection of porcine eyes from a living body results in the cessation of aqueous humor production and blood flow, programmed cell death regulated by TPR53BP2 and BCL2 genes may occur in the pigmented epithelium (PE) and non-pigmented epithelium (NPE) of the ciliary body. Blood products are subjected to gamma irradiation in order to prevent cellular damage resulting from transfusion-associated graft-versus-host disease.We investigated whether gamma irradiation influenced BCL2 or TPR53BP2 genes as well as the morphology of the porcine ciliary body. METHODS: We irradiated the anterior segments of porcine eyes by using (60)Co gamma-rays (20 Gy). To study BCL2 and TPR53BP2 expression, the irradiated specimens were fixed in formalin and embedded in paraffin and then incubated with mouse monoclonal anti-human BCL2 or TPR53BP2 antibody. RESULTS: Following dissection, an imbalance in homeostasis began with positive BCL2 and TPR53BP2 expression in the edematous ciliary processes, and resulted in atrophy of the NPE. Increased BCL2 and TPR53BP2 expression were evident just after gamma irradiation. Decreased TPR53BP2 expression occurred after 8 h of incubation, and thereby suppressed apoptosis in the NPE; hence, the structure of the ciliary body that was incubated for 8 h after gamma irradiation was well preserved. CONCLUSIONS: Irradiation renders the ciliary body in enucleated porcine eyes less vulnerable to apoptosis, and thereby exerts a profound preservative effect.