Synthesis and secretion of transferrin by isolated ciliary epithelium of rabbit

It has been shown that the vitreous contains several intrinsic glycoproteins whose origin remains to be clarified. Isolated ciliary epithelium (CE) was assayed to verify its role in the synthesis and secretion of transferrin for the vitreous body. It was cultured in the presence of [35S]methionine and the incubation medium was processed for immunoprecipitation. Total RNA from CE was processed for RT-PCR and the amplification products were sequenced. Also, whole preparations of isolated CE were processed for immunolocalization of transferrin. From the incubation assays, a labeled peptide of about 80 kDa was immunopurified that is the expected size of transferrin. The RT-PCR and sequencing experiments detected the presence of transferrin mRNA. Both layers of the CE exhibited transferrin reactivity, following immunohistochemical processing. Taken altogether, these results indicate the CE as one of the possible sources of vitreous intrinsic transferrin.     

Proliferation,differentiation and ciliary beating of human respiratory ciliated cells in primary culture

Summary The growth, differentiation, ciliary beating pattern and frequency of human respiratory ciliated cells in primary culture were studied by scanning and transmission electron microscopy and by videomicroscopy. The epithelial cells were obtained as outgrowth from explants of adult nasal polyps. When the explants were grown on type-I and type-IV collagen substrates in a standard serum-free, hormone-supplemented medium, a high percentage of ciliated cells (range 29±5% to 37±6%) was present within 2 days of culture. After 5 days of culture, the percentage of ciliated cells near the explant was 51±5%. Most of the cultured ciliated cells (85%) were characterized by individual cilia showing a coordinated movement during the beat cycle and a beating frequency (13.3±1.3 Hz) similar to that reported in vivo. In the other 15% of the ciliated cells, the dyskinetic cilia were aggregated into clumps and characterized by a rigid and planar bending movement and a lower (P<0.01) beating frequency (10.7±1.4 Hz). It is suggested that the latter type of cell, already described during fetal development, might be an intermediate type of ciliated cell which appears temporarily during the surface respiratory epithelial differentiation.     

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.     

Defining the proteome of human iris,ciliary body,retinal pigment epithelium,and choroid

The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS‐PAGE. After in‐gel digestion, peptides were analyzed using LC‐MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false‐positive rates of <0.1% and <1%, respectively. Forty‐three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four “missing proteins” were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194.     

Single modified cilia displayed by cells of human internal stratified epithelia (oral cavity,vagina)

Summary Serial sections of human vaginal and keratinized oral-gingival epithelia were investigated for ciliary structures. Most melanocytes of the gingival epithelium lacked cilia, whereas almost all basal keratinocytes of the deeper portion of the epithelial ridges possessed one cilium each. In the suprabasal layers of the ridges only a few keratinocytes exhibited a single cilium. In the basal layer, at the top of the connective tissue papillae, approximately every second keratinocyte displayed a single cilium. In the suprabasal layers above the ridges no ciliated keratinocytes were observed. The basal cells of the vaginal epithelium were endowed with cilia, while cilia were absent from the suprabasal cells. In the human forearm epidermis most melanocytes and keratinocytes are supplied with a single cilium; it has been suggested that they may play a role in light reception. However, the widespread occurrence of 9 + 0 cilia in epithelial cells of internal epithelia and their coincidence with the sites of renewal of keratinocytes suggests that a relationship may exist between solitary cilia and mitotic activity.     

Localisation of substance P-like immunoreactivity in the ciliary ganglia of monkey (Macaca fascicularis) and cat: a light- and electron-microscopic study

The present study describes substance P-like immunoreactivity in the ciliary ganglia of monkey (Macaca fascicularis) and cat. About 60% of neurons in the monkey ciliary ganglion and 40% in the cat ciliary ganglion were substance P-like immunoreactive, ranging from faint to moderate staining. Substance P-like immunoreactivity was located in cell bodies, dendritic profiles and axons. In the monkey, substance P-like immunoreactive pericellular arborisations were associated with about 0.5%–3% of the ganglion cells, which were either negatively, faintly or moderately stained. An electron-microscopic study demonstrated the presence of either substance P-like immunoreactive positive or negative axon terminals synapsing or closely associated with positive dendritic profiles in both the monkey and cat ciliary ganglia. The results suggest that substance P plays an important role in the ciliary ganglion, perhaps as a modulator or transmitter.     

A blood-aqueous barrier to small molecules in the ciliary processes of the vervet monkey (Cercopithecus aethiops)

Summary Sectors of anterior segments of vervet eyes were exposed to solutions of different osmolarities (Cercopithecus aethiops). After hypertonic incubation followed by isotonic fixation, as well as after fixation directly in hypertonic fixative, the ciliary epithelium showed constant changes. These changes consisted of a shrinkage pattern with dilations of intercellular clefts in the superficial region of the epithelium, whereas no dilations occurred in the basal layer. The basal junctional complex of the superficial cell layer was always intact. The detailed structure of this complex is described. The conclusion is drawn that it functions as a barrier to the molecules of the solutes in question, and that it may also have this function in vivo with regard to molecules of similar size.     

Secretory differentiation and cell type identification of a human fetal bronchial epithelial cell line (HFBE)

A human fetal bronchial epithelial cell line (HFBE) grew in an undifferentiated pattern under conventional culture conditions. Despite a somewhat fibro-blastic shape the cells maintained immunoreactivity to cytokeratin, carcinoembryonic antigen and epithelial membrane antigen. When grown on a collagen gel in a growth-hormone-supplemented medium, their spindle shape became more conspicuous. With an additional supplement of vitamin A (6 μg/ml), most of the cells underwent differentiation by producing many bright inclusion bodies which proved to be strongly positive with periodic acid-Schiff and weakly positive with alcian blue staining. Electron microscopy revealed a well-developed rough endoplasmic reticulum, an enlarged Golgi apparatus and many highly electron-dense secretory granules resembling those of Clara cells. Biochemical analysis demonstrated that HFBE cells cultured on collagen gel with vitamin A secreted hyaluronic acid and neutral glycoproteins containing mainly N-linked glycoproteins whose glycans were of a complex type. A monoclonal antibody (SEC-41) generated against the neutral glyco-proteins detected a glycoprotein of approximately 52 kDa in the spent culture medium of differentiated HFBE cells. This antibody also reacted with the intracytoplasmic secretory granules in these cells. When tested on frozen sections of lung tissue, the immunohistochemical reactivity of the SEC-41 antibody was confined to Clara cells, some type II pneumocytes in the adult lung, and respiratory epithelial cells in the fetal lung. More-over, this antibody could detect secretory glycoprotein in broncho-alveolar lavages from two patients. This paper clearly demonstrates that cells derived from human fetal bronchial epithelium can be cultivated in an undifferentiated precursor state and, under appropriate culture conditions, can be stimulated to undergo differentiation into a Clara cell type.     

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     

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.     

Neural stem cells in the adult ciliary epithelium express GFAP and are regulated by Wnt signaling

The identification of neural stem cells with retinal potential in the ciliary epithelium (CE) of the adult mammals is of considerable interest because of their potential for replacing or rescuing degenerating retinal neurons in disease or injury. The evaluation of such a potential requires characterization of these cells with regard to their phenotypic properties, potential, and regulatory mechanisms. Here, we demonstrate that rat CE stem cells/progenitors in neurosphere culture display astrocytic nature in terms of expressing glial intermediate neurofilament protein, GFAP. The GFAP-expressing CE stem cells/progenitors form neurospheres in proliferating conditions and generate neurons when shifted to differentiating conditions. These cells express components of the canonical Wnt pathway and its activation promotes their proliferation. Furthermore, we demonstrate that the activation of the canonical Wnt pathway influences neuronal differentiation of CE stem cells/progenitors in a context dependent manner. Our observations suggest that CE stem cells/progenitors share phenotypic properties and regulatory mechanism(s) with neural stem cells elsewhere in the adult CNS.     

Characterization, Localization, and Biosynthesis of an Interstitial Retinol-Binding Glycoprotein in the Human Eye

Abstract: Human eyes contain an M_r 135K retinol-binding protein that is analogous to interstitial retinol-binding protein (IRBP) in the subretinal space of bovine eyes. It is a glycoprotein, because it binds ¹²⁵I-concanavalin A, ¹²⁵I-wheat germ agglutinin and ¹²⁵I- Lens culinaris hemagglutinin. It does not bind Ricinus communis agglutinin I. After desialation, it binds Ricinus communis agglutinin I, but loses its capacity to bind wheat germ agglutinin. These observations, coupled with the known specificities of these lectins, suggest that at least one of the oligosaccharide chains is a sialated, biantennary complex type containing fucose. Both by direct analysis of dissected ocular tissues and by immunocytochemistry it was shown that human interstitial retinol binding protein is an extracellular protein that is confined predominantly to the subretinal space. Monkey retinas incubated in vitro in medium containing [³H]leucine were shown to synthesize and secrete this protein into the medium, a conclusion that was confirmed by immunoprecipitation with an immunoglobulin fraction prepared from rabbit antibovine IRBP serum. Virtually no other labeled proteins were detectable in the medium. It is concluded that interstitial retinol-binding protein meets many of the requirements for a putative transport protein implicated in the transfer of retinol between the pigment epithelium and retina during the visual cycle, and that the neural retina may play an important role in regulating its amount in the subretinal space.     

重组人睫状神经营养因子衍生物的构建、表达和活性分析

采用PCR定点突变技术对睫状神经营养因子(CNTF)基因进行改造,获得了CNTF衍生物(CNTF-D)基因序列分析证明其核苷酸序列符合设计要求,并在大肠杆菌中获得高效表达,CNTF-D表达量超过40%,复性后,经Q-Sepharose HP纯化,其纯度超过95%,体内法测定发现能明显降低实验小鼠的体重。表明CNTF-D在体内具有良好的生物学活性,为下游开发奠定了基础。     

Effects of reversible nare occlusion on the development of the olfactory epithelium in the rabbit nasal septum

Summary To investigate environmental influences on the development of the olfactory epithelium, semi-thin sections were taken from the nasal septum of newborn and 30-dayold rabbits; the epithelial thickness and the number of olfactory knobs, supporting cells, dark basal cells, and receptor cells were compared. During normal development, a marked increase in epithelial thickness was found, largely because of an increase in the number of receptor cells. Whereas unilateral nare occlusion on day 1 resulted in 10% fewer receptor cells and 25% fewer knobs on the deprived side by day 30, nare occlusion either up to or after day 5 had little effect, and even temporary reopening from days 6–7 was sufficient to stimulate receptor-cell development on the occluded side. Although in these latter cases, a slight deprivation effect of 6% was still found in the number of receptor-cell nuclei, there was no longer a significant difference in the number of knobs between the open and closed sides. Thus, whereas exposure to the environment during the first days of life appears to be sufficient to stimulate sustained growth, the deprived epithelium may retain the capacity to respond to such cues beyond this time. However, as nare occlusion also had an effect on the respiratory epithelium and nasal lymphatic tissue, the nature of the cues stimulating receptor-cell development, whether olfactory or non-olfactory, is not yet clear.     

Cholinergic nerves stimulate mucociliary transport,ciliary activity,and mucus secretion in the frog palate

Summary Mucociliary transport, ciliary activity, and mucus secretion were studied in the palate of the frog Rana pipiens by direct observation, stroboscopic synchronization of ciliary beating, and histochemistry. Excised palates were studied in vitro, and intact palates were studied in vivo. Electrical stimulation of the glossopharyngeal nerve in vivo or of the palatine nerve in vitro stimulated all three activities. The effect was mimicked by acetylcholine and pilocarpine, enhanced by physostigmine, and blocked by atropine but unaffected by d-tubocurarine. Stimulation increased the number of cilia beating and their rate of beating, the number of goblet cells secreting and, for small acidic cells, the amount of mucus secreted, and the rate and extent of particle transport. The response to tactile stimulation was locally restricted in vitro but widespread in vivo. It was concluded that, although there is a low basal rate of mucus secretion and ciliary activity that is independent of nervous control, stimulation of these activities in the intact animal is mediated through the central nervous system and cholinergic nerves to the palate.Supported in part by Grant HL-16730 from the U.S. Public Health Service     

Identification of a Neuropeptide and Neuropeptide-Processing Enzymes in Aqueous Humor Confers Neuroendocrine Features to the Human Ocular Ciliary Epithelium

Abstract: The ocular ciliary epithelium, the site of aqueous humor secretion in the mammalian eye, is believed to play a key function in signaling mechanisms that regulate the rate of secretion, and thus intraocular pressure. One possible way of mediating these signaling functions is through neuropeptides and hormones secreted into the aqueous humor and acting on target tissues. We recently identified a cDNA clone sharing 100% identity with carboxypeptidase E (CPE), a neuropeptide-processing enzyme. Utilizing polymerase chain reaction, we further identified and characterized another processing enzyme, the peptidylglycine α-amidating monooxygenase (PAM), and the neuropeptide secretogranin II, a molecular marker restricted to neuroendocrine tissues. Using specific probes, we found that the nonpigmented ciliary epithelial cells express CPE, PAM, and secretogranin II mRNA, and protein. We also found that CPE and secretogranin II are abundant in aqueous humor. Treatment of cultured ciliary epithelial cells with veratridine and phorbol ester up-regulates CPE and PAM. Secretogranin II was found to be induced by veratridine, whereas phorbol ester had little effect, suggesting different mechanisms for secretion. The results demonstrate that secretogranin II, CPE, and PAM represent a specialized group of neuropeptide and neuropeptide-processing enzymes secreted by the ciliary epithelial cells which may confer to them neuroendocrine functions in cell-cell communication or cell signaling.     

A new approach to pharmacological effects on ciliary beat frequency in cell cultures—exemplary measurements under Pelargonium sidoidesextract (EPs 7630)

The ciliary beat frequency (CBF) is an important parameter of the defence mechanism of the mucociliary system. We present a new method to determine pharmacological effects on CBF in vitro. Ciliated cell cultures of human nasal epithelium were obtained from partial resection of hyperplastic inferior turbinates in rhinosurgery. An adherent monolayer culture of ciliated cells was present after 10 days in vitro. This study exemplary examines, if a special extract from the roots of Pelargonium sidoides (EPs 7630) has an effect on the CBF in vitro. The influence of three concentrations of the extract (1, 30, 100 microg/ml) was tested. EPs 7630 significantly and concentration-dependently increased CBF to 123% at 30 microg/ml and to 133% at 100 microg/ml compared to the equilibration phase (100%). After rinsing with extract-free medium the CBF of cultured cells returned to nearly the normal range. In future, drug manipulation of the CBF by local application of rhinologics could be a new therapeutical concept in the treatment of upper airway diseases.     

Studies on the distribution of phosphorylase in eyes of the rabbit and the squirrel monkey

Summary Detailed studies on phosphorylase localization in various components of the eyeball of rabbit and squirrel monkey have been made. Corneal epithelium, endothelium, and stromal cells, extrinsic and intrinsic muscles of the eyeball, ciliary process, endothelial cells of the anterior chamber angle, vitreal cells, lens epithelium, inner segment of cones; plexiform layer, ganglion cell layer, internal and external limiting membrane and Muller cells show high phosphorylase activity. Surprisingly, we observed phosphorylase activity inside the nucleus in the posterior 2 to 3 layers of corneal epithelium. The significance of phosphorylase localization in relation to glycogen distribution in various components of the eyeball and their energy requirements is stressed.