In order to contrast anionic sites in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules. After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl2 concentration used during staining. At 0.4 M MgCl2, the length was mostly within the range 100-180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.
Several connective tissues were stained for proteoglycans using the cationic dye Cuprolinic Blue according to the critical electrolyte concentration method. With this method, proteoglycans are visualized as electron-dense filaments. In most tissues, two types of proteoglycan filaments are present: a small (maximum length 60 nm), thin, collagen fibril-associated filament, and a thick, heavily-staining filament which is predominantly localized between bundles of collagen fibrils. Cartilage contains very large (about 300 nm) proteoglycan filaments while in cornea they are very small. Comparison with biochemical data from the literature suggests that the appearance of the proteoglycan filaments may be indicative for the glycosaminoglycan-protein ratio and for the molecular weight of the part of the protein core to which glycosaminoglycans are attached. The data thus obtained on the localization and structure of a proteoglycan may be useful when planning a strategy for its isolation.
The nature of Cuprolinic Blue-positive anionic filaments in mouse lung alveoli has been characterized. The contrast of filaments in the alveolar basement membrane of type I epithelial cells was lost on treatment with nitrous acid and pronase (without prefixation). In contrast, neither neuraminidase, chondroitinase ABC or AC, nor Streptomyces hyaluronidase had any effect. Treatment with pronase (after prefixation) and 2.0 M MgCl2 (after prefixation) also had no effect, indicating that the filaments are heparan sulphate proteoglycans. The filaments in the alveolar basement membrane of type II epithelial cells and in the capillary basement membrane of the endothelial cells were also nitrous acid sensitive, but chondroitinase ABC-insensitive. A model in which the whole alveolus contains a single layer of heparan sulphate-containing proteoglycan monomers is proposed. Furthermore, the collagen fibril associated filaments remained unaffected after treatment with nitrous acid, neuraminidase or Streptomyces hyaluronidase, or after digestion with pronase (after prefixation) and treatment with 2.0 M MgCl2 (after prefixation). These filaments, however, could no longer be detected when digestion with chondroitinase ABC or pronase (without prefixation) was applied; chondroitinase AC treatment clearly affected the filaments, although they still were visible. These results indicate that the filaments are dermatan sulphate-containing proteoglycans. Some functional aspects of the proteoglycans are discussed.
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