A comparison of membrane enzymes of human and pig oesophagus; the pig oesophagus is a good model for studies of the gullet in man

Summary The distribution and relative catalytic activities of five plasma membrane enzymes (alkaline phosphatase, dipeptidyl peptidase IV, γ-glutamyl transpeptidase, microsomal alanyl aminopeptidase and glutamyl aminopeptidase) were examined in human and pig oesophagus. In both species, alkaline phosphatase activity occurred in basal and suprabasal cells of the epithelium and in capillaries. Stromal cells in the human submucosa were particularly reactive. Dipeptidyl peptidase IV was present in blood vessels and capillaries in man and pig and in submucous glands in the pig. The enzyme was also present in both species in the lamina propria cells immediately adjacent to the epithelial basal lamina. In the human, γ-glutamyl transpeptidase occurred in the epithelial basal cells and in isolated basal and lower prickle cells in the pig. Stromal cells in the human submucosa were strongly reactive and capillaries in the muscularis propria in both species moderately active. Microsomal alanyl aminopeptidase was detected in lamina propria cells adjacent to the epithelial basal cell layer in man and pig and at the apices of mucous cells in pig submucous glands. Weak glutamyl aminopeptidase activity was confined to capillaries in both species. The findings of this study, along with the ready availability of pig oesophagus, suggest that the pig may be a suitable model for studies of the gullet in man.     

Orthogonal arrays in normal and injured respiratory airway epithelium

Orthogonal arrays are found on plasma membranes of glial cells, in the central nervous system, on muscle plasma membranes at neuromuscular junctions, and on a variety of epithelial cells. These structures have been correlated with ion flux. With the aid of freeze fracture technique, orthogonal particle arrays were found on plasma membranes on airway epithelial cells of rats and hamsters. They have been found in abundance at the base of secretory cells throughout normal airway epithelium. These structures were found to increase in number during regeneration in response to injury and they were found in great numbers on plasma membranes of all airway cells in response to acute and chronic NO2 exposure. The lateral and basal plasma membranes of the respiratory epithelium are a new source for studying orthogonal arrays. The normal number and distribution of these arrays can be perturbed in response to mechanical and chemical injury.     

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.     

The ability of an epithelium to survive removal of the basal lamina by enzymes: fine structure and content of sodium and potassium of the midgut epithelium of the larva of Tenebrio molitor after withdrawal of the basal lamina by elastase--a short note

A method is described to separate the epithelial cells of the posterior part of the mealworm midgut from their thick basal lamina using elastase. After removal of the basal lamina the naked epithelial cells remain connected with each other, still forming a midgut tube. The ultrastructural changes observed are enlargement of the lateral spaces between the cells and simultaneous destruction of junctional structures other than desmosomes and tight junctions. This enlargement is most probably due to shrinkage of the epithelial cells as a consequence of osmotic stress, which the cells normally seem to be protected against by the basal lamina. The content of sodium and potassium in the epithelium is not influenced by the elastase treatment, indicating that the midgut tube stays alive with intact plasma membranes.     

Hemidesmosomes of normal and regenerating mouse corneal epithelium

Hemidesmosomes of normal mouse corneal epithelium observed in tangential thin sections, occupy 14% of the basal plasma membrane. They consist of linear chains of densities with an orientation that is not random with respect to the radial axis of the cornea, tending to parallel it. During the repair of a small epithelial defect, cells of the corneal epithelium peripheral to the defect show chains of hemidesmosomes arranged parallel to the direction of migration of the epithelial sheet. This is parallel to the radius, like the orientation of the normal chains. Cells of the area that was denuded of epithelium, and is being resurfaced, show no hemidesmosomes. During repair of a large defect of the corneal epithelium hemidesmosomes are present on the cells covering the denuded area but they are small, few in number compared to the normal, and many are not arranged in chains. These small hemidesmosomes appear to be points of attachment of very fine basal filaments, possibly actin.     

Comparative study of a new ultrastructural specialization of the mammalian retinal pigment epithelium: basal intracytoplasmic tubules

We recently described basal intracytoplasmic tubules that arise from the basal plasma membrane of rat retinal pigment epithelial cells (RPE). They are a previously undescribed ultrastructural specialization of the basal plasma membrane in addition to the well-known basal folds. This report describes similar tubules in the RPE cells of the rabbit, hamster, and kitten. As in the rat, the tubules in the hamster and kitten ramify singly through the basal cytoplasm and bear no special relationship to any other organelle. In the rabbit, however, stacks of closely apposed tubules frequently abut the large lipid droplets in their RPE cells. Although the function of the tubules is unknown, their occurrence in several different mammals suggests they are involved in the function of RPE cells in general.     

Ultrastructure of the capillaries of nerve tissue transplants growing in the anterior chamber of the eye

Blood capillaries have been studied electron microscopically in the areas of grafts (rat embryonal hippocamp and septal cerebral parts transplanted to mature rats) containing mainly nervous, glial or connective tissue cells. Certain differences in the capillary wall structure have been revealed. In areas with a great concentration of nervous cells, the blood capillaries are characterized by a dense arrangement of cellular elements in their walls, a continuous layer of the glial end-feet, this is specific for the CNS capillaries providing the blood--brain barrier. In peripheral area of the grafts, where glial elements predominate, the capillaries have loose arrangement of the mural cellular elements, great endotheliocyte activity, thick connective tissue tunic, lack of a dense glial surrounding. These characteristics make dubious the statement whether these capillaries possess the blood--brain barrier function. In places where connective tissue cells make aggregates, the capillaries do not possess the barrier properties because of perforations and fenestrae in endothelium and interruptions of the basal membrane, absence of pericapillary glial elements. All types of the capillaries demonstrate certain signs of a high functional activity. Formation of the capillary structure depends on the surrounding tissue.     

Fine structural observations of the pecten oculi capillaries of the chicken

Summary The pecten oculi of the domestic chicken was examined with light, scanning and transmission electron microscopy and with freeze-etching techniques. Particular attention has been given to the capillary structure. The capillaries form an extensive anastomotic network. Their endothelial cells have apical (luminal), as well as basal, longitudinally oriented microfolds. It is assumed that the formation of apical differentiations of the endothelial surface is due to haemodynamic influences. Thus, sufficient surface area for membrane bound enzymes is achieved. These enzymes are necessary for active transcellular transport processes that require energy. In freeze-etched material, two different structures of the membrane surface of microfolds can be recognized. These results are discussed in relation to transport functions through capillary endothelial cells of the pecten. It is assumed that the pecten plays an important role in the nourishment of the retina and vitreous body. This paper was presented in part at the inaugural session of the European Club for Ophthalmic Fine Structure in Essen on January 20 and 21, 1973.     

Synthesis and migration of 3H-fucose-labeled glycoproteins in the retinal pigment epithelium of albino rats, as visualized by radioautography

3H-fucose was injected into the vitreous body of the eye(s) of 250-gm rats, which were then killed by means of an intracardiac perfusion with glutaraldehyde after intervals of 10 min, 1 and 4 hr, and 1 and 7 days. The eyes were removed and further fixed, and pieces of retina were processed for light and electron microscope radioautography. Light microscope radioautography showed that the pigment epithelial cells actively incorporated 3H-fucose label. The intensity of reaction peaked at 4 hr after injection of the label and then slowly declined. Quantitative electron microscope radioautography revealed that, at 10 min after 3H-fucose injection, over 70% of the label was localized to the Golgi apparatus, indicating that fucose residues are added to newly synthesized glycoproteins principally at this site. With time the proportion of label associated with the Golgi apparatus decreased, but that assigned to the infolded basal plasma membrane, the apical microvilli, and various apical lysosomes increased. These results indicate that in retinal pigment epithelial cells newly synthesized glycoproteins continuously migrate from the Golgi apparatus to lysosomes and to various regions of the plasma membrane. In this case, the membrane glycoproteins may play specific roles in receptor functions of the basal plasma membrane or phagocytic activities at the apical surface. Very little label migrated to Bruch's membrane, indicating either a very slow turnover or a paucity of fucose-containing glycoproteins at this site.     

Alkaline phosphatase, 5'-nucleotidase and magnesium-dependent adenosine triphosphatase activities in the transitional epithelium of the rat urinary bladder.

The cerium-based method was used to demonstrate cytochemically the ultrastructural localization of alkaline phosphatase (ALPase), 5'-nucleotidase (5'-Nase) and magnesium-dependent adenosine triphosphatase (Mg-ATPase) on the transitional epithelium of the rat urinary bladder. The reaction product for ALPase was found on the plasma membrane of all epithelial cells, except the luminal surface of superficial cells. The activity of 5'-Nase appeared on the plasma membrane of all bladder transitional epithelial cells, including the free surface of superficial cells. The Mg-ATPase reaction product was seen on the plasma membrane of superficial, intermediate and basal cells, but never on the luminal surface of superficial cells and it was only occasionally seen on the basal surface. The possible functions of these phosphatases have been discussed, and it was emphasized that the 5'-Nase activity present on the luminal surface of superficial cells may play a special role in the membrane movement of these cells in the transitional epithelium.     

Plasma membrane-associated vesicles in retinal capillaries of the rat

The structure and function of abluminal vesicles in endothelial cells of rat retinal capillaries was examined using glutaraldehyde-tannic acid fixation and the hemeproteins--horseradish peroxidase, microperoxidase, and lactoperoxidase--as tracers. Numerous vesicles, delimited by a tannic acid-positive membrane, were distributed along the abluminal front. Other vesicles were arranged in clusters and chains or tubule-like structures. Such vesicles were not found in the vicinity of the capillary lumen. When the retina was exposed to hemeproteins, either in vitro or after intravitreal injection, the abluminal vesicles became labeled with tracer reaction product. Apparently "free" vesicles and tubules seen in tangential sections through the basal lamina were also labeled, suggesting that they were in continuity with the plasma membrane in another plane of section. No enzyme reaction product was present in the capillary lumen. Peroxidase-positive multivesicular bodies were observed, suggesting that some protein was endocytosed and directed to lysosomes where it was presumably degraded. The results suggest that abluminal endothelial vesicles represent pits or invaginations of the plasma membrane and, as such, are not involved in the transendothelial transport of protein from the perivascular space to the capillary lumen. Tannic acid treatment revealed a population of similar vesicles associated with the plasma membrane of pericytes. After exposure to hemeproteins, enzyme reaction product was localized in these vesicles and in a few multivesicular bodies. The results suggest that the majority of these vesicles are in continuity with the plasma membrane and are not involved in endocytosis.     

Absorption of horseradish peroxidase by the small intestinal epithelium in postnatal developing rats.

After an intraluminal injection of horseradish peroxidase into the small intestine, the localization of peroxidase was studied in neonatal developing and adult rats by means of electron microscopy. Until around the 14th day of the neonatal period absorbed peroxidase granules in the duodenal and jejunal epithelium were abundant in the microvillous membrane, the apical tubulo-vacuolar system, and the Golgi apparatus, and on the lateral cell and basal membranes, and the luminal surfaces of the capillary cells. At the weaning period the tubulo-vacuolar system was absent in the duodenal and jejunal epithelial cells, and at that point absorbed peroxidase was observed in the same sites as in the adult rats: the microvillous membrane, the lateral cell and basal membranes, the Golgi apparatus, and the vesicles and vacuoles of the cytoplasm. During the suckling period, in the ileal epithelial cells exogenous peroxidase was found on the microvilli, in the tubulo-vacuolar system, in the supranuclear vacuole, in the Golgi apparatus, on the lateral cell and basal membranes, and also on the luminal surface of the endothelial cells of blood capillaries. When the tubulo-vacuolar system and the supranuclear vacuole were lost from the ileal cells at the weaning period, no exogenous peroxidase uptake was observed in the absorptive cell of the ileal epithelium.     

Cytological observations of the ovarian epithelium in mammals during the reproductive cycle

We have studied the ovarian epithelium at various stages of the reproductive cycle in a number of mammalian species utilizing light microscopy, scanning microscopy, the freeze-fracture technique, transmission microscopy and by employing specialized tracers that use lanthanum and horseradish peroxidase. We found that the epithelial cells are joined by incomplete tight junctions, gap junctions, and desmosomes. The cytoplasmic matrix contains a large irregularly shaped nucleus, few microtubules, microfilaments, mitochondria, endoplasmic reticulum and a host of coated and non-coated vesicles of varying diameters. The saccules comprising the large Golgi complex and its companion vesicles are associated with a basal body-centriole complex: some of these saccules and affiliated vesicles are acid phosphatase positive. Surface modifications of ovarian epithelial cells include numerous microvilli, some of which have a bulbous tip, and plications of the lateral plasma membrane which are thought to accommodate volume changes of the ovary during follicular development. Many coated and non-coated endocytotic caveolae were found on these cells, particularly in the basal area. These caveolae internalized exogeneously administered horseradish peroxidase. We view the marked endocytotic activity as an efficient transport mechanism for partially removing substances from the interstitium of the ovary and the peritoneum.     

The serine/threonine kinase dPak is required for polarized assembly of F-actin bundles and apical-basal polarity in the Drosophila follicular epithelium

During epithelial development cells become polarized along their apical-basal axis and some epithelia also exhibit polarity in the plane of the tissue. Mutations in the gene encoding a Drosophila Pak family serine/threonine kinase, dPak, disrupt the follicular epithelium that covers developing egg chambers during oogenesis. The follicular epithelium normally exhibits planar polarized organization of basal F-actin bundles such that they lie perpendicular to the anterior-posterior axis of the egg chamber, and requires contact with the basement membrane for apical-basal polarization. During oogenesis, dPak becomes localized to the basal end of follicle cells and is required for polarized organization of the basal actin cytoskeleton and for epithelial integrity and apical-basal polarity. The receptor protein tyrosine phosphatase Dlar and integrins, all receptors for extracellular matrix proteins, are required for polarization of the basal F-actin bundles, and for correct dPak localization in follicle cells. dpak mutant follicle cells show increased beta(Heavy)-spectrin levels, and we speculate that dPak regulation of beta(Heavy)-spectrin, a known participant in the maintenance of membrane domains, is required for correct apical-basal polarization of the membrane. We propose that dPak mediates communication between the basement membrane and intracellular proteins required for polarization of the basal F-actin and for apical-basal polarity.     

Topographic features of the organization of lymphatic capillaries and lipid resorption in the jejunal villi of the white rat

As demonstrate serial semithin sections and transmissive electron microscopy, there is not one but a group (2-7) of lymphatic capillaries with anastomoses between them in the villus of the white rat jejunum. In the superior parts of the villus the lumen in the lymphatic capillaries is maximal, and their distance to the epithelial basal membrane of the anterior and posterior surfaces is small. In the inferior part of the villus, when the size of the lumen in the lymphatic microvessels is minimal, the greatest distance between them and the basal membrane of epithelium covering the mentioned surfaces of the villus is noted. In the superior and middle parts of the villus paracellular transport of lipids from the interstitial space into the lumen of the lymphatic capillaries predominate, in the inferior part--transcellular is the main way of transport. The topographic peculiarities of the lymphatic microvessels in the superior and middle parts of the villus make, in combination with the active paracellular transport, the morphological basis of a more intensive absorbtion of lipids from the intestinal lumen.     

Rhombic particle arrays in gill epithelium of a mollusc, Aplysia californica.

Gill epithelia from adult and juvenile Aplysia were examined by conventional thin section and freeze-fracture methods. Freeze-fracture replicas of adult gill epithelium revealed septate and gap junctions, which served as membrane markers for the epithelial cells. In these same cell membranes, non-junctional rhombic arrays of intramembranous particles were observed on prominent ridges on the membrane P fracture face of some epithelial cells. In thin sections of adult epithelium, nerve terminals were observed abutting the lateral plasma membranes near the basal lamina of some epithelial cells. Correlative areas of plasma membrane in freeze-fracture replicas showed a close association between rhombic particle arrays and abutting nerve terminals. In thin sections of juvenile Aplysia, nerve terminals abutting the epithelial cells were not recognizable, and rhombic arrays were not observed in freeze-fracture replicas. This suggested that a developmental association existed between the appearance of rhombic arrays in adult epithelia and their innervation. It is not known with certainty if, in invertebrates, rhombic arrays are an essential structural entity of all innervated cell membranes; however, in the cells thus far studied, there appears to be an associative condition. In the case of the gill epithelium of Aplysia, rhombic arrays are located in the same vicinity as the abutting nerve terminals. Similar arrays of intramembranous particles have been observed in myoneural postjunctional complexes of other invertebrates and have been interpreted to be the morphological expression of neurotransmitter receptors. An analogous explanation is put forth, namely that rhombic arrays may represent the structural correlates of neurotransmitter receptors and/or ionic channels in innervated membranes of invertebrates.     

Immunocytochemical localization of the glucose-transport protein in mammalian brain capillaries

Summary The endothelial cells of mammalian brain capillaries, which form the anatomical basis of the blood-brain barrier, have been investigated by immunocytochemical methods to determine the distribution of the glucose-transport protein. A monoclonal antibody raised against the intact human erythrocyte glucose-transport protein and polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal sequence of the human erythrocyte glucose-transport protein were used for immunofluorescent staining of isolated human and bovine cerebral cortex microvessels. The pattern of fluorescence with both antibodies demonstrated the antigen to the distributed throughout the plasma membrane of the capillary endothelial cells. These results provide further evidence for the homology between the human erythrocyte and brain capillary glucose-transport protein, and confirm its abundance in brain capillaries.     

Effects of melittin on a model renal epithelium, the toad urinary bladder

The bee venom melittin, 10(-6) M, on the mucosal (urinary) side of the toad urinary bladder (in vitro), markedly decreased transepithelial potential difference, short-circuit current (Isc, sodium-dependent) and resistance. However, these effects were not seen when the toxin was placed on the opposite (serosal) side of the membrane preparation. The electrical effects were accompanied by a large increase in the transepithelial permeability to 22Na. The response was not changed by meclofenamic acid (which blocks formation of prostaglandins) but it was inhibited by La3+. In the presence of amiloride, which usually inhibits active Na transport and Isc, melittin, on the mucosal side, increased the Isc. The action of melittin appears to involve an interaction with anionic sites, which mediate its effects. Such sites appear to be present on the apical plasma membranes of the toad bladder epithelial cells, but they are not as abundant or they are inaccessible on the basal plasma membrane.     

Cationic dyes reveal proteoglycans on the surface of epithelial and endothelial kidney cells

Summary The glomerular epithelial cells of the rat kidney fixed by vascular perfusion with an aldehyde solution containing either safranine O or alcian blue (and 0.3 M MgCl₂) display filaments which are located close to the outer surface of the plasma membrane. These filaments are similar to those revealed by the same methods in the laminae rarae of the glomerular basement membrane. Alcian blue (and MgCl₂) further demonstrates the presence of anionic sites inside the endothelial cell pores of the glomerular and peritubular capillaries, on the luminal surface of endothelial cells of large renal vessels and along the basolateral surface of the epithelial cells of the Bowman capsule and of the proximal convoluted tubule.Supported in part by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 146)     

Morphological observations on the unique paired capillaries of the opossum retina

Light-microscopic and ultrastructural analysis of the ocular tissues of the North American opossum (Didelphis virginiana) revealed that the arterial and venous segments of retinal vessels, including capillaries of the smallest calibre, occur in pairs. They do not form anastomotic networks, the common pattern in mammals with vascularised retinae, but instead the two segments of the pair join to form hairpin end loops. The pairedd vessels, with the arteriolar limb usually on the vitread aspect, penetrate the retina and branch to form three distinct layers of capillaries. The most superficial lies in the nerve fiber layer, the middle is situated in the inner nuclear layer and the deepest extends to the external limiting membrane, which is considerably deeper than in normal mammalian holangiotic retinae. The paired capillaries display classical morphological features of central nervous system capillaries, i.e., they are lined by continuous endothelial cells united by tight junctions. The lining endothelium is supported by a distinct basal lamina that splits to envelop pericytes. The latter, although abundant, are invariably interposed between the two vessels that form each vascular unit. Phylogenetic and functional aspects of this unique form of retinal vascularisation are discussed.