Isolation of the basal and lateral plasma membranes of rat kidney tubule cells.

A method was developed to isolate renal basolateral membranes from cortical kidney tubule cells of single rats. The isolated membrane fraction was characterized by the measurement of marker enzyme activities and by electron microscopy. 1. After centrifugation of crude plasma membranes on a discontinuous sucrose density gradient the basolateral membranes accumulated at a sucrose density of p= 1.14-1.15 g/ml. The yield was 147 mug membrane protein/g kidney wet weight. Protein recovery was 0.1%. 2. (Na+ + K+)-ATPase was enriched 22-fold from the homogenate. The recovery was 2.6%. The (Na+ + K+)/Mg2+-ATPase ratio was 4.1. 3. The contamination by brush borders was small. Alkaline phosphatase was 1.6-fold enriched and 0.2% was recovered. Aminopeptidase was 1-fold enriched with a recovery of 0.1%. The contamination by mitochondria, lysosomes and endoplasmic reticulum was negligible. 4. In electron micrographs the basolateral membranes showed a typical triple layered profile and were characterized by the presence of junctional complexes, gap junctions or tight junctions.

Morphological aspects of potassium flow in the semicircular canal ampulla of the pigeon.

Potassium ions are a prerequisite for the development and regulation of sensory cell stimulation in the inner ear. From the potassium-rich endolymph the ions flow into the sensory cells apically and are released basolaterally. After transport pathways of various lengths potassium is released again into the endolymph - in the cochlea by marginal cells of the stria vascularis, in the vestibular labyrinth by dark cells. While this long recycling pathway is relatively well-known in the cochlea, few studies have been conducted on the semicircular canal ampullae (SCCA) where its morphological basis is largely unknown. According to the present electron microscopic findings, potassium ions are initially released into the extracellular space during stimulation of the sensory cells and then absorbed by supporting and light cells. Finally they are transported transcellularly over numerous very long gap junctions into the region of the dark cells. From here they move to an extracellular compartment, which is more or less completely sealed off basally by basal plates of the light cells. Apically the intercellular space between light and dark cells is sealed by junctional complexes. This newly identified space in the SCCA corresponds to the extracellular compartment between the marginal and intermediate cells in the stria vascularis. At both sites, the cochlea and the SCCA, this probably serves as a regulatory valve, reservoir or storage space, particularly for potassium ions. It is likely that the different morphology of the ion transport pathways is related to the different flow levels of potassium ions expressed by the different levels of the so-called endocochlear potential and concomitant movement of other ions in the cochlea and SCCA.

The morphology of basement membrane formation.

A transplantable, basement-membrane (BM)-producing murine tumor was investigated using immunofluorescence and electron microscopy. Collagen type IV, fibronectin, laminin, and a BM proteoglycan (BM-1) could be discriminated in the intercellular substance (ICS). The occurrence of these substances and the electron microscopic findings speak for a comparability of the ICS of the tumor with the BM of normal tissues. Hence, a model has become available to study the morphology of BM-formation. The BM-producing cells are characterized by numerous, often enlarged cavities of the rough endoplasmic reticulum (RER) and a large Golgi complex. The BM-material is released into the ICS via prosecretion and electron-dense secretion granules. The morphology of the extracellular BM-material depends upon the fixation technique. After addition of tannic acid, typical BM-lamellae are formed, which in most cases show a granular structure. This is due to shrinkage and attachment of glycoproteins to the collagen skeleton. After ruthenium red or after glutaraldehyde fixation, the extracellular matrix consists of homogeneously distributed filamentous material with only few granular structures. This situation corresponds to the small amount of acid proteoglycans in the BM. The present findings facilitate the evaluation of BM-formation in other cell types.

The influence of colchicine on the cytotoxic effect of hydroxyurea in the embryonic spinal cord of the mouse.

The development of necroses and the course of the mitotic index in the embryonic spinal cord of 10-day-old mouse embryos (NMRI) were investigated between 1.5 h and 4 h after either an intraperitoneal application of 500 mg/kg hydroxyurea (HU) or 1 mg/kg colchicine or a simultaneous application of both substances. One h and 30 min after colchicine application, the mitotic index of the neuroepithelial cells had decreased (5.22%; control 7.7%). Subsequently, the mitotic index increased, reaching a maximum of 10.35% 2 h and 30 min after colchicine application. Between 1 h and 30 min and 4 h after HU application, the mitotic index decreased from 4.6% to 0.61% (control 7.7%). First necroses of neuroepithelial cells were observed 1 h and 50 min after HU application. 4 h after HU treatment, 34.38% of the neuroepithelial cells were necrotic. After simultaneous application of HU and colchicine, the decrease of the mitotic index was similar to the decrease after HU application alone. No necroses were observed in the embryonic spinal cord during a 4 h period after simultaneous application of HU and colchicine. After colchicine, as well as after simultaneous HU and colchicine application, the shape of the nuclei of the neuroepithelial cells changed from oval to round and the chromatin was condensed. Mechanisms for the inhibition of the cytotoxic but not the cytokinetic effects of HU by colchicine are discussed.

Basement membrane formation and lung cell differentiation in vitro.

In high density cultures of mouse fetal lung cells, so-called "mass cultures", development of organoid structures, formation of a basement membrane (BM), and differentiation of pneumocytes type II occur accompanied by synthesis and secretion of lamellar bodies. The relationship between the formation of a BM, on the one hand, and morphogenesis as well as differentiation of pneumocytes type II, on the other hand, has been investigated by use of antibodies against BM components in the lung mass culture. It is shown here that anti-laminin antibodies prevented BM formation, but morphogenesis and pneumocyte differentiation occurred as in untreated cultures. Short-term treatment with the antibody revealed that the BM is formed only during the first 2 to 3 days in vitro. Already formed BM could not be removed by anti-laminin. Anti-collagen type IV antibodies showed no effect in the lung mass culture except for a stronger staining of the BM. Anti-BM-1 antibodies caused no changes in morphogenesis, cell differentiation and BM formation either, but the mesenchymal intercellular space exhibited a dark staining, which is probably due to antigen-antibody complexes. The results obtained with anti-laminin antibodies indicate that a BM is not necessary for lung cell differentiation in vitro.

Pre- and postnatal development of the primary visual cortex of the common marmoset. II. Formation, remodelling, and elimination of synapses as overlapping processes.

During ontogenesis changes in the numerical density of synapses are usually assumed to depend essentially on variations in the formation of synapses. Only the final adjustment to adult synapse densities is thought to include the elimination of synapses in some brain regions of certain species. Here, we focus attention on quantitative aspects of synapse elimination throughout development of area 17 of marmoset monkeys (Callithrix jacchus). Mature synapses, various precursor forms, and indicators of lysosomal degradation of synapses were quantitatively analysed by electron microscopy and morphometric methods. A total number of about 135 x 10(9) synapses was calculated for area 17 in each adult hemisphere corresponding to a volume density of 600 x 10(6) synapses/mm3. At 3 months of age, the respective values were 508 x 10(9)/area and 1,159 x 10(6)/mm3, while at birth these values were 69 x 10(9)/area and 328 x 10(6)/mm3. Consequently, at least three out of four synapses are eliminated between 3 months and adulthood. However, the real number of synapses being eliminated during development is probably much larger if the time course of lysosomal degradation is additionally taken into account. The frequency of lysosomes in presynaptic endings is highest before net-elimination of synapses occurs, i.e., between 1 and 3 months. This suggests that lysosomal degradation is not directly responsible for the majority of synapses removed during ontogenesis but apparently represents a second mechanism for synapse remodelling and elimination. Thus, it appears from this study that remodelling and elimination of synapses are quantitatively as important as their formation, and accompany synaptogenesis from its very onset onwards.

Pre- and postnatal development of the primary visual cortex of the common marmoset. I. A changing space for synaptogenesis.

The primary visual cortex of Callithrix jacchus occupies a large portion of the occipital neocortex and can be safely delineated from fetal stages onwards. In 20 animals ranging in age from fetal to adult age the morphological development of area 17 was evaluated and compared with the growth of whole brain, skull, and head size. Cortical thickness, surface area, and volume of the area were determined in addition to predominant growth directions. The volume of area 17 approximately doubles between birth (241 mm3) and three months of age (506 mm3). This maximum value marks an overshoot in growth (volume: 180%, surface area: 150%, thickness: 122%), which is followed by a considerable reduction before adult values (100%) are reached. Although these values seem to indicate that the overall reduction in size is fairly isometric, growth and regression are locally anisometric. For example, layers II-IVc contribute disproportionately to the overshoot; thickening is less pronounced than tangential growth and follows a slightly different time course. These data suggest that the developing visual cortex represents a highly dynamic distribution space for the developing synaptic junctions which should be taken into account in studies on synaptogenesis. By comparison it is suggested that this growth dynamic is not restricted to area 17 but also occurs in some other parts of the cerebral cortex. In contrast, most subcortical brain regions apparently do not undergo overshoot growth. Structural changes of the skull compensate the overshoot in cortex growth, so that head size increases steadily.

Changes in integrin expression during chondrogenesis in vitro: an immunomorphological study.

Integrins are receptors composed of ligand-specific alpha-chains and cell type-specific beta-chains which are involved in cell-cell and cell-matrix interactions. The distribution of alpha 1- and alpha 3-integrins as well as collagen Types I and II, was investigated by immunofluorescence and immunoelectron microscopy during chondrogenesis in organ culture after various culture periods. Mesenchymal cells from limb buds of Day 12 mouse embryos were grown at high density. Within the first 2 days of the culture period, only alpha 1-integrin could be detected. Formation of cartilage-specific matrix on Day 3 was accompanied by the occurrence of alpha 3-integrin. On Day 7, alpha 3 was present only in cartilage nodules, whereas alpha 1 was strongly expressed in the perichondrium and was more or less homogeneously distributed in the surrounding mesenchyme. On Day 14, alpha 1-integrin was again detectable in cartilage. We suggest that the change in collagen formation from Type I to Type II during chondrogenesis is accompanied by a change in integrin expression from alpha 1 to alpha 3. Conversely, dedifferentiation of chondrocytes in aging cartilage is accompanied by the occurrence of collagen Type I and alpha 1-integrin. Therefore, a strict correlation between the collagen type synthesized by the cells and the appropriate receptor presented by the cells is suggested.

Development and properties of malignant lymphoma induced by magnesium deficiency in rats.

Female wistar rats were fed on an Mg-deficient diet. After 8 to 10 weeks, the thymus glands were strongly degenerated and the number of lymphocytes was reduced, especially in the cortex. Thymus degeneration was associated with a decreased rate of DNA, RNA, and protein biosynthesis and necrosis and phagocytosis of lymphocytes. The degeneration was normalized after feeding on an Mg-rich diet. After 10 or 11 weeks of Mg deficiency, local cell proliferations of immature lymphocytes with a great number of free ribosomes were found in some thymus glands. The local cell proliferations developed into infiltrating tumors without metastases. In lymphoma cells the Na+ and Ca2+ content, the turnover of cellular Na+ and K+, and the aerobic production of CO2 and lactate were increased.

The thyroid gland of Callithrix jacchus in organ culture.

Thyroid glands from 7 marmosets (Callithrix jacchus) of different age groups (newborn, 2 weeks, 2, 7, 8 and 11 months, and 8 years) were grown as organ culture according to Trowell at the medium/air interphase for 3 to 30 days. The morphology of thyroid tissue was well preserved until the end of the culture period. Necroses were only occasionally seen in connective tissue. In contrast to the in vivo situation, the number of lysosome-like inclusions changed considerably and cell-cell contacts became loose. After the addition of TSH the number of apical microvilli, apical granules and coated vesicles rose. The basal surface of follicle cells enlarged due to the formation of processes or ridges and invaginations. Mitosis could be observed. These findings show that, using the Trowell culture technique, thyroid tissue can be grown for up to 4 weeks without showing distinct changes in its morphology. Its responsiveness to the application of TSH is maintained in vitro. Hence, this technique is suitable also for long-term endocrinological, pharmacological and toxicological investigations.

The influence of matrix components on the morphological differentiation of a proliferating hepatocyte line from liver of newborn mice.

The differentiation behaviour of a liver epithelial cell line of the newborn mouse cultured on various matrix components (Thermanox pure, Thermanox coated with ECM, dried collagen type I and type II, wet collagen type I and type III and on floating collagen) was investigated by electron microscopy. Only during the last few days of pregnancy and up to day 9 p.p. could these cells be isolated using a very delicate method. The cells were smaller than differentiated hepatocytes and proliferated spontaneously. They resembled the so-called oval liver cells. On Thermanox pure or Thermanox coated with ECM, dried collagen type I or type II a confluent monolayer developed after about 6 days that consisted of rather flat extended cells which were characterized by short contacts and the absence of any morphological indications of differentiation. On wet collagen the extension area was smaller and the cells were taller. The length of the contact area and the number and size of gap junctions and cell organelles increased. On floating collagen multi-layered aggregates of polygonal cells developed that were characterized by extended cell contacts, bile capillary-like structures and highly developed cell organelles, especially rough endoplasmic reticulum. Since differentiation processes can be demonstrated ultrastructurally only on wet collagen, especially on floating collagen, the chemical composition of the substrate and a specific matrix-cell interaction cannot be the only triggering factor. It is assumed that mechanical properties of the substrate, e.g. plasticity, are involved. The change in the shape of the cell, the prolongation or intensification of the cell contact and the adaptation of the cytoskeleton might play a decisive role in this connection.

Electron microscopic observations on the formation of elastic fibres in cultures of aortic medial cells and adventitial cells.

The formation of elastic fibres was observed in the cultured cells derived from the tunica media and the tunica adventitia of mouse aorta. Bundles of myofilaments with dense bodies were abundantly observed in the cytoplasm of the cultured medial cells, and numerous bundles of microfibrillar components were present in the intercellular spaces. Fine granules of approximately 50 nm in diameter were observed in the bundles of microfibrillar components. It was supposed that these fine granules of elastin fused with each other and formed elastic aggregates and then formed large elastic clumps. Numerous bundles of microfibrillar components were also present in the intercellular spaces of the cultured adventitial cells. Elastic aggregates were scarcely observed in the bundles of microfibrillar components. However, large elastic clumps as observed in the medial cell culture could not be found in the adventitial cell culture. It is suggested that the formation of large elastic clumps might be related to the sheet structures or lamellae of elastic fibres in the tunica media.

Ultrastructural changes in the synovial membrane in experimentally-induced osteoarthritis of rabbit knee joint.

Rabbit knee joint osteoarthritis was induced by intraarticular injections of a 10% sterile NaCl solution. Within 30 days the synovial membrane had undergone hyperplasia resulting in activated synovial fibroblasts. Transitional forms of synoviocytes as well as activated synovial macrophages were a very common finding. At 60 days a thickening of the synovial intima was perceptible. Most of the synoviocytes were of the fibroblast type. Transitional cell forms abounded. An increase in collagen fibres and capillaries of the fenestrated type occurred in the intercellular spaces. In the deep layer collagen bundles had formed between which activated fibroblasts and macrophages were noticed. The described changes point to an active participation of the synovial membrane in the destruction of articular cartilage in osteoarthritis.

Matrix changes during long-term cultivation of cartilage (organoid or high-density cultures).

In high density (organoid or micromass) cultures of prechondrogenic mesenchymal cells from limb buds of 12-day-old mouse embryos typical cartilaginous tissue develops after 3 days. Immunomorphological investigations have shown that it contains the typical components of the cartilaginous matrix, such as collagen type II and cartilage-specific proteoglycans. After a 2-week cultivation period hypertrophic cartilage cells develop to an increasing extent. Many of these cells as well as normal chondroblasts detach from the matrix from the 2nd week in vitro onwards to assume a fibroblast-like appearance. At the same time thick (25-65 nm) collagenous fibrils occur at the surface of these cells. These thick fibrils contain collagen type I, as shown by immunomorphology. Hence, in these older cartilage cultures chondroblasts change their synthesis programme or direction of differentiation. Consequently, a model for the study of "dedifferentiation" of cartilage and possibly also transformation of cartilage cells to osteoblasts has become available.

Integrins in ageing cartilage tissue in vitro.

Matrix-cell interactions are of great importance for numerous cell functions whereby integrins play an essential role as transmitters of extracellular signals. In cultures of ageing cartilage tissue (organoid or high density cultures) cartilage cells occur on the surface of which thick fibrils of collagen type I are deposited. Since integrins, in their role as receptors, cause an interaction between matrix components and cell membrane, we tried to demonstrate immunomorphologically (light and electron microscopically) the corresponding integrin receptors for collagen type I (beta 1 alpha 1 and beta 1 alpha 2) on the surface of these ageing cartilage cells. Cultures of normal, i.e. young cartilage tissue exhibit only beta 1 alpha 3- and beta 1 alpha 5-receptors; labelling against the integrins beta 1 alpha 1 and beta 1 alpha 2 is not possible in this case. Our results show that after the occurrence of thick fibrils cartilage cells express new receptors (beta 1 alpha 1 and beta 1 alpha 2) on the cell membrane. Thus, in ageing or dedifferentiating cartilage tissue it is not only the synthesis programme of matrix components (e.g. instead of collagen type II >> collagen type I) which changes but also the integrins (instead of alpha 3/beta 1, alpha 5/beta 1 >> alpha 1/beta 1, alpha 2/beta 1) so that new collagen types can be bound. These findings may also serve for a better understanding and interpretation of cartilage changes in vivo during ageing and under pathological conditions.

Co-localization of integrins and matrix metalloproteinases in the extracellular matrix of chondrocyte cultures.

Beta1-integrins were found in the cartilage matrix, suggesting their implication in the assembly of its architectural scaffold, but the mechanism for this event is not yet clear. Matrix metalloproteinases (MMPs) may be involved in an integrin-shedding mechanism and matrix beta1-integrins may act to alter MMP activity. To begin to address this question, this study was designed to determine whether beta1-integrins and MMPs are colocalized in the chondrocytes or in the extracellular matrix of cartilage. We investigated high-density cultures of limb buds of 12-day-old mouse embryos by double immunofluorescence, immunoelectron microscopy and by coimmunoprecipitation assays in order to examine the localization of beta1-integrins and matrix metalloproteinases (MMP-1, MMP-3 and MMP-9) in cartilage. It was found, that all investigated MMPs and beta1-integrins were specifically co-localized in high-density cartilage cultures. Immunogold and immunofluorescence labelling of both beta1-integrins and MMPs were observed not only at the surface of chondrocytes but mainly also in the pericellular space and distributed between collagen fibrils in the extracellular matrix (ECM) as well. Results of immunoprecipitation experiments suggest a functional association of MMPs and beta1-integrins in chondrocytes as already described for other cell types. Further investigations are needed to elucidate the functional association between beta1-integrins and MMPs in chondrocytes.

Morphology, differentiation and matrix production of liver cells in organoid cultures (high density cultures) of fetal rat livers.

The aim of this study was to demonstrate the morphology and matrix synthesis of embryonic rat liver cells (day 18 of gestation) in organoid cultures (high density cultures) with electron microscopic and immunomorphological techniques. For this purpose the cells of embryonic rat livers were isolated enzymatically and grown in an organoid culture (high density culture) for 3 weeks in a Trowell system. During the first 48 h a sorting-out process took place, i.e. liver and blood-forming cells met to form aggregates. In between mesenchymal cells were seen. Vessel-like cavities developed. Electron microscopic inspection of the hepatocytes did not reveal any lesions of the cell organelles after 14 days in culture. As late as after a 3-week culture period mitochondrial swellings and an increased number of autophagic vacuoles were observed. A rim of collagenous fibrils or fibrillar bundles and granular matrix structures was perceptible as early as after 7 days in culture. Immunofluorescence microscopic techniques revealed collagen types III, IV and VI as well as laminin, nidogen, heparansulfate-proteoglycan and fibronectin in these areas. Thus, the composition of the matrix in this culture system corresponds (apart from the absence of collagen type I) to the embryonic situation. Therefore, the organoid culture appears to be an appropriate technique to study the behaviour of hepatocytes in vitro. It is especially suited to demonstrate the formation of matrix components in liver cells and their extracellular occurrence.

The morphology of mesangial cells cultured at high density and in collagen gels.

Primary mesangial cells (rat) from monolayer cultures of the 6th to 12th passage and permanent SV40 Mes13 cells were grown at high density in organoid culture at the medium/air interphase. After adaptation to the in vitro conditions, both mesangial cell types developed after 7 days a synthesis apparatus (endoplasmic reticulum, Golgi apparatus) and produced matrix which consisted of Lamina densa-like material, collagenous fibrils and filaments. Unspecific contacts, gap junctions and adhesion belts could be demonstrated in the contact areas. Additionally, some cells exhibited thick bundles of actin filaments. A close resemblance of the mesangial cells in high density culture to those in vivo can, therefore, be stated. Hence, they differentiated with regard to their matrix formation, contraction and contact behaviour and can therefore be used for experimental studies within a short culture period of 7 days. Cell aggregates in monolayer culture and in cultures in collagen gels had not differentiated at this stage.

Connective tissue of the livers of newborn and adult marmosets (Callithrix jacchus).

Immunofluorescence microscopic and electron microscopic investigations revealed components of the matrix and of the basal lamina (collagen type I, III, IV and V, BL-heparan sulfate and fibronectin) in the sinus wall (Disse's space) of the livers of newborn and adult marmosets (Callithrix jacchus). Collagen type I was missing in both the two age groups. Small amounts of laminin were present in the livers of newborn and absent in those of adult animals, whereas collagen type III occurred in the form of delicate fibres. Light microscopic inspection showed a continuous distribution of all other components in the sinus wall. The amount of collagen type III and V increased depending on the age. Electron microscopic investigations revealed single or bundled fibrils (20-30 nm) and filaments (10-12 nm). After addition of tannic acid, plaques of a fine-filamentous network and incorporated granules were observed. After addition of resting Ruthenium Red, electron-dense granules (20-60 nm) were irregularly distributed in the structureless space, resting on collagenous fibrils and cell membranes. The fibrils were allocated to collagen type III, the filaments to collagen type V. The plaques were supposed to contain heparan sulfate, collagen type IV and fibronectin. The absence of a Lamina densa of the basal lamina was attributed to the absence of laminin which probably plays an important role in the formation of this layer. Differences in the distribution pattern of the matrix components and thus a functional mosaic of the permeability of Disse's space were assumed. The complete absence of collagen type I and laminin in the lobules makes the adult marmoset liver especially suited for studies on the importance of this collagen type under pathological conditions, since both components are expressed in this way.

Electron microscopic studies of ion- and H2O-transporting epithelial cells in the horizontal ampulla of the pigeon.

Earlier morphological studies of the epithelial structure in the semicircular canals of mammals have focused on the sensory cells of the crista ampullaris. This report draws attention to the fact that there exist at least seven further cell types in the horizontal ampulla walls of pigeon with various functions; the role of ion- and H2O-transporting epithelial cells is dealt with here in detail. While the dark cells appear to play a decisive role in the regulation of ionic composition, the cells in the planum semilunatum may transport H2O and assist in the regulation of endolymph volume. In addition, protein-secreting structures are located in the apical region of the cells of the planum semilunatum. The question whether the proteins are dispersed in the endolymph or contribute to cupula formation remains unclear. The morphology and possible functions of these two cell types are discussed on the basis of electron microscopic results.