Asymmetric localization of Notch2 on the microvillous surface in choroid plexus epithelial cells

Notch family molecules are transmembrane receptors that play various roles in contact-dependent cell–cell interactions in a wide range of organs. In the brain, Notch2, but not the other members of Notch, is expressed in the choroid plexus at an exceptionally high level. We immunohistochemically examined the cellular and subcellular localization of Notch2 protein in the choroid plexus using confocal and electron microscopy. Unexpectedly, Notch2 was asymmetrically localized on the microvillous surface of epithelial cells in the choroid plexus of both postnatal and adult rats. This localization pattern of Notch2 suggests its novel and unknown role independent of contact with adjacent cells in the choroid plexus. In organotypic cultures of the choroid plexus, the addition of anti-Notch2 antibody resulted in deformation of microvilli in epithelial cells, which suggests a role of Notch2 in the maintenance of the microvillous structure in choroid plexus epithelial cells.     

gamma-Glutamyl transpeptidase in rat liver during 3'-Me-DAB hepatocarcinogenesis: immunohistochemical and enzyme histochemical study

Immunohistochemical localization of gamma-glutamyl transpeptidase (gamma-GTP) in rat liver during 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) hepatocarcinogenesis was investigated and compared with sites of gamma-GTP activity. Immunohistochemically, gamma-GTP was stained in the apical border of proliferating oval cells during the early stages of azo-dye carcinogen feeding. After 7 weeks, multiple hyperplastic nodules appeared in which gamma-GTP was localized in the bile canaliculi. In hepatoma tissues, positive staining for gamma-GTP was observed in the bile canaliculi-like spaces, on the cell membrane, and sometimes in the cytoplasm of malignant cells. Enzyme histochemical staining showed gamma-GTP activity to be present in almost the same areas as the immunoreactive gamma-GTP. However, some areas adjacent to hepatoma tissue showed immunohistochemically reactive protein but no enzyme activity. Immunoreactive gamma-GTP was present in all locations at which enzyme activity was seen. The present data suggest that an altered form of gamma-GTP might be present in tissues during 3'-Me-DAB hepatocarcinogenesis.     

A marker of acid-secreting membrane movement in rat gastric parietal cells

A monoclonal antibody (mab 146.14) marker of the movement of acid-secreting membranes in rat gastric parital cells has been produced and characterized. Mab 146.14 recognized a 95-kD major component of a purified membrane fraction of rat gastric mucosa, the protein composition of which was similar to that of well characterized porcine H+ -K+ ATPase-enriched membranes, and that presented the characteristic shift of density depending on whether it was purified from resting or stimulated tissues. Further biochemical analysis characterized the antigen as a membranous protein that might be in its native form, part of a higher multimolecular complex. Immunocytochemical localization of the antigen demonstrated that only membranes related to acid secretion in parietal cells expressed the 95-kD antigen. In resting conditions, the 95-kD antigen was diffusely distributed in the cell cytoplasm associated with inactive tubulovesicles. In stimulated cells, by contrast, all the antigen was recovered associated with secretory active microvilli formed by the apical insertion of the previously resting internal tubulovesicles. In conclusion, the 95-kD antigen, presumably a part of the rat gastric proton pump, is a marker of acid-secreting membranes in rat parietal cells. The translocation of antigen and membranes, observed by both light and electron microscopy supports the fusion model of membrane insertion from a cytoplasmic storage pool to the apical surface upon stimulation of acid secretion.     

A novel mechanism for group translocation: substrate-product reutilization by gamma-glutamyl transpeptidase in peptide and amino acid transport.

Gamma-glutamyl transpeptidase (gamma-GTP) is suggested to act as a carrier in the group translocation of oligopeptides and possibly some amino acids across cellular membranes. It is proposed that the process may involve the repetitive transfer of gamma-glutamyl groups to acceptor peptides which are being translocated from the exterior of the cell to its interior. After group translocation of the peptides has occurred with concomitant formation of gamma-glutamyl peptide products, it is suggested that the products might then be utilized as substrate for the enzyme in order to permit the translocation of other peptides from the exterior. The system is economical and requires only that it be primed with an appropriate source of gamma-glutamyl peptides, such as glutathione. In contrast to most group translocation systems previously described, substrate-product reutilization by gamma-GTP would not be expected to accumulate peptides against a concentration gradient. Mechanisms for maintaining low intracellular concentrations of the translocated peptides are described. Studies on acceptor substrate specificity of gamma-GTP from bovine choroid plexus and rat kidney show some glycyl peptides are much better substrates than free amino acids in accord with the proposal that gamma-GTP might be primarily involved in peptide translocation. Both kinetic and topological evidence support the suggestion that repetitive transfer of gamma-glutamyl moieties by gamma-GTP could occur during group translocation of peptides and possibly some amino acids.     

Retinal-specific ATP-binding cassette transporter (ABCR/ABCA4) is expressed at the choroid plexus in rat brain

ATP-binding cassette (ABC) transporter A4 is a member of the ABC transporter subfamily A which has been reported to be exclusively expressed in the retina. In contrast, a previous report has suggested a possible relationship between ABCA4 and CNS function. The purpose of the present study was to investigate the localization of ABCA4 mRNA and protein in rat brain. In situ hybridization analysis revealed that ABCA4 mRNA was localized in the lateral ventricles. RT-PCR analysis detected ABCA4 mRNA in isolated rat choroid plexus and conditionally immortalized rat choroid plexus epithelial cells (TR-CSFB). Furthermore, ABCA4 protein was also detected in the isolated rat choroid plexus at about 250 kDa by western blot analysis, and its apparent molecular size was reduced by N-glycosidase F treatment. These results suggest that glycosylated ABCA4 protein is expressed in rat choroid plexus epithelial cells. ABCA4 may play a role in the function of the blood-cerebrospinal fluid barrier and affect CSF conditions.     

Ultrastructural localization of carbonic anhydrase activity in the rat choroid plexus epithelial cell

Summary Localization of carbonic anhydrase activity was studied electron microscopically on cells of the rat choroid plexus epithelium. For the ultracytochemical detection of these activities, Yokota's technique (1969), which is the modification of Hansson's method (1967) was employed. Numerous electron dense reaction products were observed in the microvilli of the choroidal epithelial cell. The reaction deposits were also remarkably present in the infoldings of the basal plasmalemma but to a lesser extent than in the microvilli. The localization sites were mainly on the plasma membrane, but some reaction products were also observed in the cytoplasm near the plasma membrane. Hardly any reaction product was found in the intracellular organelles except for the mitochondria in which reaction products were occasionally observed on the cristae. These activities were completely inhibited by acetazolamide. As the carbonic anhydrase activity was histochemically seen in the microvilli and the basal infoldings, it is likely that carbonic anhydrase is related to an active transport process in the secretion of cerebrospinal fluid as is Na⁺, K⁺-ATPase (Masuzawa et al. 1980).     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na⁺/K⁺-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

Glycogen phosphorylase activity and immunoreactivity during pre- and postnatal development of rat brain

Catalytic activity and immunoreactivity of glycogen phosphorylase were studied in pre- and postnatal rat brain. The catalytic activity was assayed in brain homogenates; immunoreactivity was investigated by immunoblot analysis using a monoclonal anti-bovine brain glycogen phosphorylase antibody. The cellular localization and intensity of immunoreactivity were analysed on paraffin-embedded sections utilizing the same monoclonal antibody. The catalytic activity increased 10-fold from embryonic day 16 to adult; immunoreactivity became detectable on embryonic day 16 and increased in intensity as the enzyme activity rose to adult values. The first cellular elements to be stained immunohistochemically were ependymal cells lining the ventricles, ependymal cells of the choroid plexus, meningeal cells and a selected population of neurons in the brain stem. The immunoreactivity of plexus cells and meningeal cells was reduced or absent in the adult rat brain. The earliest appearance of glycogen phosphorylase immunoreactivity in astroglial cells was seen at postnatal day 9 in the hippocampus. The staining pattern of the adult brain was reached at day 22 post partum. The developmental changes in glycogen deposition and in glycogen phophorylase activity and immunoreactivity may indicate a variable physiological role of glycogen metabolism for different cell types in the pre- and postnatal periods.Dedicated to Professor Helmut Leonhardt on the occasion of his 75th birthday     

Expression of nerve growth factor receptor immunoreactivity in the rat choroid plexus.

The presence and localization of nerve growth factor receptors (NGFr) in the choroid plexus of the adult rat has been investigated immunohistochemically using an anti-rat NGFr monoclonal antibody (192-IgG). A moderate to strong immunoreaction was observed in the epithelial cells of the choroid plexus, whereas the choroidal blood vessels and connective tissue remained unlabelled. Moreover, no sex-differences were encountered in the NGFr immunoreaction intensity and Bouin fixative was more effective than 10% formaldehyde evidenciating the NGFr immunostain. Occasionally, ependymal cells displaying NGFr immunoreactivity were observed. Present data demonstrate that the choroid plexus of the rat contain NGFr, probably low-affinity NGFr, and suggest an involvement of NGF in the regulation of cerebrospinal fluid secretion, but the importance of these findings, if any, must be investigated in future studies.     

Localization of osteopontin in resorption lacunae formed by osteoclast-like cells: a study by a novel monoclonal antibody which recognizes rat osteopontin

The characteristics of a monoclonal antibody produced against osteoclast-like multinucleated cells (MNCs) formed in rat bone marrow cultures were examined immunohistochemically and biochemically. The in vitro immunization was performed using as immunogen the MNCs from rat bone marrow cell culture, which revealed many characteristics of osteoclasts. After screening and cloning of hybridomas, the monoclonal antibody HOK 1 was obtained. This antibody reacted weakly with stromal cells and intensely with both MNCs and their putative migratory traces on culture dishes. Immunofluorescent examination of paraffin sections revealed intense reactivity on the epithelium of the choroid plexus, the ileum and the proximal-convoluted tubules of the kidney, and also on bone cells such as osteocytes, osteoblasts, and osteoclasts. Western blotting using purified rat osteopontin verified that the antigen recognized by HOK 1 was osteopontin. Positive HOK 1 immunoreactivity was further observed in the resorption lacunae formed by a culture of MNCs on human tooth slices and on the surface of osteoclasts. The present data suggested that osteopontin is preferentially present on the resorption lacunae in resorbing calcified matrices and that osteoclasts under a specific state might trap this protein on their cell surface.     

Immunocytochemical localization of Na+,K+-ATPase catalytic polypeptide in mouse choroid plexus

Na+,K+-ATPase plays a central role in the mechanism of cerebrospinal fluid secretion by the choroid plexus. We have used an antiserum to the 100 KD catalytic polypeptide of the enzyme purified from mouse brain (30) to localize the catalytic unit in mouse choroid plexus at the light and electron microscopic levels. Pre-embedding immunostaining with the peroxidase-conjugated second antibody technique showed that microvillar borders facing the ventricle were intensely reactive. In contrast, basal and lateral plasma membrane surfaces were devoid of activity. Identical localization was obtained with a post-embedding procedure in which protein A-gold was used to stain immunoreactive sites on thin sections of Lowicryl-embedded tissue. For comparison, immunogold staining was shown to be restricted to basolateral membranes of kidney medullary ascending thick limbs. The apical localization of Na+,K+-ATPase in choroid plexus is in striking contrast to the almost exclusive basolateral localization seen in other ion-transporting tissues. The immunocytochemical data are completely consistent with physiological data on choroidal epithelial transport and with light microscopic autoradiographic localization of [3H]-ouabain binding sites.     

Differential binding patterns of three antibodies (VOBM1, VOBM2, and VOM2) in the rat vomeronasal organ and accessory olfactory bulb

Immunohistochemical properties of monoclonal antibodies raised against the rat vomeronasal epithelium were examined in adult rats. Three monoclonal antibodies, VOBM1, VOBM2, and VOM2, reacted specifically to the luminal surface of the sensory epithelium of the vomeronasal organ. In addition, the reactivities of VOBM1 and VOBM2 were detected in the vomeronasal nerve layer and the glomerular layer of the accessory olfactory bulb. Electron-microscopic study revealed differential patterns of the immunoreactivity of the three antibodies to the microvilli of vomeronasal sensory epithelium. VOBM1 immunoreactivity was found on the microvilli of the supporting cells, whereas VOBM2 immunoreactivity was found on those of the sensory cells. VOM2 immunoreactivity was observed on the microvilli of both the sensory and supporting cells. These results suggest that the three antibodies recognize different antigens on the vomeronasal sensory epithelium. In particular, VOBM2 antibody appears to react to an antigen specific to the microvilli of the vomeronasal sensory cells.     

Monoclonal antibody to a common antigen of secretory granule membranes: intracellular localization and recycling of the antigen after secretion.

Monoclonal antibody (MAb) 170-5 was generated to the secretory granule membrane of rat parotid acinar cells. The MAb recognized integral membrane glycoproteins (SG 170 antigen) localized on the luminal side of the secretory granules with N-linked carbohydrates, molecular weights 92, 84, 76, 69, and 65 KD. Immunohistochemical studies indicated that the SG 170 antigen was found in the secretory granules of both exocrine and endocrine cells and in the lysosomes of various cells in the rat. Immunoelectron microscopy with immunogold revealed that the antigen was present on the membrane of the secretory granules, lysosomes, the Golgi vesicles, and condensing vacuoles in pancreatic and parotid acinar cells and in AR42J rat pancreatic tumor cells; the Golgi stacks exhibited no immunoreaction. The common localization of the antigen in the secretory granule membranes indicated that this antigen may play an essential role in regulated secretion. Employing HRP-labeled MAb 170-5, we followed the retrieval of the antigen after exocytosis in AR42J cells. The MAb was internalized specifically with antigen-mediated endocytosis. It was transported to endosomes, subsequently to the trans-Golgi network, and then packaged into secretory granules. However, the Golgi stacks revealed no uptake of the labeled antibody.     

Immunohistochemical demonstration of glycogen phosphorylase in rat brain slices

Paraffin-embedded sections from paraformaldehyde-fixed rat brain were stained immunocytochemically for glycogen phosphorylase brain isozyme BB, using a monoclonal mouse antibody and the biotin-strept-avidin method, with either horseradish peroxidase or beta-galactosidase as marker enzymes. Two cell types showed strong glycogen phosphorylase-immunoreactivity: Astrocytes and ependymal cells. Most intensive staining was observed in the cerebellar cortex, the neocortex and the hippocampus. Astrocytes in the cerebellar white matter stained positively. The choroid plexus cells stained poorly or not at all. Neurons throughout the brain were negative, as well as oligodendrocytes and bundles of myelinated nerve fibers. These data are consistent with the immunocytochemical localization of glycogen phosphorylase in astroglia-rich primary cultures derived from rat brain.     

Expression and characterization of alkaline phosphatases during differentiation of human pancreatic cancer (Capan-1) cells in culture.

Human pancreatic cells of the Capan-1 cell line differentiate in culture. During the exponential growth phase, the cells are undifferentiated, only becoming differentiated during the stationary phase. The formation of domes in this phase is related to the exchange of water and electrolytes. The present study was designed to characterize the localization and expression of alkaline phosphatases (AP) in Capan-1 cells during growth in culture. Biochemical, cytoenzymatic and immunocytochemical methods were employed combined with light and electron microscopic examination. AP essentially of the placental type were expressed progressively during the exponential growth phase, and were seen to be distributed over the surface of the Capan-1 cells. In the stationary phase, the AP became localized on the surface of microvilli. The precipitates of the enzyme reaction highlighted regular four-bodied structures. Biochemical assays showed a progressive increase in activity of this enzyme in cells during both the exponential and stationary growth phases. However, in the stationary phase between days 7 and 8, there was a fall in enzyme activity, with a corresponding increase in this activity in the culture medium. Cytological examination indicated that this fall could be accounted for by loss of AP-positive membranes by vesiculization of apical microvilli and release of microvesicles into the culture medium. Immunoblots showed that Capan-1 cells expressed two types of AP, a placental type (70 kDa) and to a lesser extent a liver type (80 kDa). Expression of the placental type was attributed to a neoplastic derepression of the coding gene, while the liver type was assumed to be a normal gene expression of human duct cells. The placental type AP might thus serve as a marker of transformation, and the liver type as a marker of differentiation.     

Abnormal embryonic development induced by antibodies to rat visceral yolk-sac endoderm: isolation of the antigen and localization to microvillar membrane

An antigenic substance was isolated from rat visceral yolk-sac endoderm of the 18th-20th days of gestation by extraction with the nonionic detergent Nonidet P-40, Sephacryl S-300 gel filtration, and Ricinus communis agglutinin affinity chromatography. The rabbit antiserum directed against this antigenic substance when injected into pregnant rats during the period of organogenesis caused abnormal embryonic development, fetal growth retardation, and embryonic death. Ouchterlony gel diffusion analysis demonstrated that the antiserum formed one immunoprecipitin band against the crude detergent extract and a complete identity between the present visceral yolk-sac antigen and the renal glycoprotein antigen previously isolated (C. C. K. Leung, (1982) J. Exp. Med. 156, 372-384). The antigen eluted from the antibody affinity column appeared to consist of two major peptides of 60 and 30 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Indirect immunofluorescent and immunoperoxidase localization studies at the light microscopic level demonstrated that both rat renal proximal tubule and embryonic visceral yolk-sac endoderm at various gestational stages (including the organogenetic period) shared the same antigen. Indirect immunoperoxidase localization studies at the electron microscopic level demonstrated that the antigen was a part of (or associated with) the microvillar membrane and membrane invaginations at the base of the microvilli of the renal proximal tubule and visceral yolk-sac endoderm. In vivo immunoperoxidase localization studies demonstrated that the teratogenic antibodies localized within the large phagolysosomes and the apical vesicles of the visceral yolk-sac endoderm. It is postulated that visceral yolk-sac pathology was induced by the antibodies.     

Alkaline phosphatase activity of the IVth ventricular choroidal epithelium of rats during embryonic and neonatal development

The cytochemical localization of alkaline phosphatase (AlPase) activity in the developing IVth ventricular choroidal epithelium was investigated in embryonic and neonatal rats. During the initial development of the choroidal primodium the flattened and/or cuboidal epithelial cells of the ventricular roof were changed to columnar cells with well-developed microvilli and apical tight junctions. When compared to AlPase activity on the lateral plasma membranes of the surrounding ependymal cells, these columnar cells of the choroidal primodium revealed activity on the lateral and luminal plasma membranes, but no activity was found on the basal surface of these cells. On the other hand, the epithelial cells in the neonatal choroid plexus showed a continuous morphological alteration from columnar cells with short microvilli to mature cuboidal cells with numerous long microvilli. AlPase activity in immature columnar cells was observed on all plasma membranes, except for the apical junctional area of the lateral surface. With maturing of the choroidal epithelial cells, the activity appeared to be eliminated from the lateral and luminal plasma membranes of the cuboidal cells, and mature choroidal epithelial cells showed activity on the basal surface only. These findings suggest that AlPase may play an important role in the membrane activity of epithelial cells differentiating between the primitive epithelial cells of the ventricular roof and the mature choroidal epithelial cells.     

Expression of the β-subunit isoforms of the Na,K-ATpase in rat embryo tissues,inner ear and choroid plexus

Summary— We report evidence of the apical localization of the two Na, K-ATPase β-subunit isoforms in cells of the inner ear and of the choroid plexus of the rat. To this end, we generated isoform-specific antisera against the human Na, K-ATPase β1 and β2 subunits. These polyclonal rabbit antisera were raised against truncated β-isoform proteins that were made in E coli with pET expression vectors. Deglycosylation of the native antigen with N-endoglycosidase F shows four bands in the β1 isoform and five bands in the β2 iso-form immunoblots. In E15 rat embryos, the β1 isoform was detected in brain, heart and kidney and the β2 isoform only in brain. While β-subunit mRNA expression (Watts AG, Sanchéz-Watts G, Emanuel JR, Levenson R 1991 Proc Natl Acad Sci USA 88, 7425–7429), and immunoblotting and enzymatic activity have been determined (Zlokovic BV, Mackic JB, Wang L, McComb JG, McDonough A 1993 J Biol Chem 268, 8019–8025), very little is known about the specific localization of each β-isoform in the epithelia of choroid plexus and inner ear. Immunocytochemical preparations of 15-day-old whole rat embryos and adult rat brain showed an enhanced staining for the β1 and β2 isoforms in the apical membrane of the ampullary crests of the inner ear's semicircular ducts and in the cuboidal cells of the choroid plexus     

Alpha-enolase is restricted to basal cells of stratified squamous epithelium.

We have developed a monoclonal antibody against a 50-kDa protein that binds preferentially to basal cells in the limbus of rat, rabbit, and human corneas (J. D. Zieske, G. Bukusoglu, and M. A. Yankauckas, Invest. Ophthalmol. Visual Sci. 33, 143-152, 1992). Here we report on the purification and identification of the antigen. The 50-kDa antigen was purified from rabbit limbal and corneal epithelium using HPLC methodology including anion exchange (DEAE) followed by reverse-phase (C18) chromatography. The purified 50-kDa protein was then digested with endoproteinase Lys-C, and a reproducible profile comprising approximately 20 peptides was observed by reverse-phase HPLC of the digest. Sequence analysis of five peptides ranging in length from 4 to 20 residues revealed that the 50-kDa protein was alpha-enolase, a glycolytic enzyme. Overall, 57 amino acids were identified with a 95% sequence homology. Localization of alpha-enolase in rat epithelium by immunofluorescence microscopy demonstrated that simple epithelium contained low or undetectable levels of the enzyme. Stratified squamous epithelium, however, showed high levels of alpha-enolase, which was localized specifically to cells of the basal layer. Epidermal, corneal limbal, oral mucosal, vaginal, and laryngeal epithelium all showed cytoplasmic binding specific to the basal cells. These data indicate that the glycolytic enzyme alpha-enolase is preferentially localized in the basal cell layer of stratified squamous epithelium and suggest that glycolytic activity is concentrated in these cells. The localization pattern suggests that a major change in metabolism occurs as cells leave the mitotically active basal cell layer and migrate toward terminal differentiation in the suprabasal cell layers.