Aleksander Hinek

Secretion of proteoglycans by chondrocytes: Influence of colchicine, cytochalasin B, and β-d-xyloside

Chondrocytes obtained from epiphyseal cartilage of fetal guinea pigs or ear cartilage of young rabbits were cultured in monolayer. The influence of colchicine, cytochalasin B, and p-nitrophenyl-β-d-xylopyranoside on secretion of proteoglycans was investigated. Radioactive sulfate was used as a precursor. As observed previously in other systems, β-d-xylosides initiated the synthesis of free chondroitin sulfate chains, competing with the endogenous proteoglycan core protein acceptor. The molecular weights of the chondroitin sulfate chains synthesized both on the xyloside and on the core-protein acceptor in maximally stimulated cells were similar and significantly lower than in proteoglycans synthesized in the absence of xyloside. The size of the chondroitin sulfate chains synthesized on the xyloside was inversely related to the concentration of this compound. This finding suggests that the chain length is dependent on the ratio between available acceptor and chain-lengthening enzymes or precursors. Cytochalasin B, a microfilament-modifying agent, inhibited proteoglycan synthesis, without any effect on secretion. Cells treated with cytochalasin B could be stimulated with β-d-xyloside to synthesize free chondroitin sulfate chains to the same relative degree as cells with intact microfilaments. Colchicine, an antimicrotubular agent, partially inhibited synthesis and secretion of proteoglycan. However, cells treated with colchicine could be stimulated with β-d-xyloside to synthesize and secrete free chondroitin sulfate chains to about the same relative degree as cells with intact microtubules. The data suggest that microtubules may have a facilitatory rather than an obligatory role in the secretion of proteoglycans and that at least part of the effect of colchicine is located at or after the site of glycosaminoglycan synthesis.

Electron microscopic and cytochemical studies of rat aorta. Intracellular vesicles containing elastin- and collagen-like material

SynopsisSmall, rounded vesicles with a dense core of amorphous material were observed in all cell types in the young rat aorta, that is, endothelial cells, smooth muscle cells and fibroblasts. They were particularly numerous in the Golgi complex but were also found in the cell periphery. The content of the vesicles had staining characteristics identical to those of elastin. Material of the same type was also found in cisternae on the maturing side of the dictyosomes and in vesicles budding from them. Reaction product for thiamine pyrophosphatase was present in both these structures, indicating that the Golgi complex is responsible for the formation of the dense-cored vesicles. This was further supported by the absence of reaction product for acid phosphatase in the cisternae and in the vesicles. Moreover, no uptake of exogenous markers was noted in the latter. On the basis of these findings it is suggested that the dense-cored vesicles have a secretory function and contain precursors of elastin.Elongated vesicles or profiles containing collagen fibrils were observed in smooth muscle cells and fibroblasts. In the cell periphery, these vesicles were often found to communicate with the extracellular space. Further inside the cells, they showed a close spatial relationship to the Golgi complex. Neither thiamine pyrophosphatase nor acid phosphatase activity was demonstrated in the elongated vesicles. Like the plasma membrane, their limiting membrane was positively stained for alkaline phosphatase. On the basis of these findings and the absence of uptake of exogenous markers in them, it is suggested that the elongated vesicles represent a means for collagen secretion in the growing aortic wall. The Golgi complex is believed to be involved in the transfer of collagen to these vesicles.

Electron microscopic observations on the formation of elastic fibers in primary cultures of aortic smooth muscle cells

Smooth muscle cells were enzymatically isolated from the tunica media of the aorta of 5-day-old rats and grown in culture for 4–28 days. A confluent monolayer was rapidly formed and the cells later grew in multiple overlapping layers. Throughout the period of observation the cells maintained the structure of vascular smooth muscle with numerous myofilaments and a well-developed granular endoplasmic reticulum and Golgi complex. An intercellular matrix with plenty of elastic fibers accumulated already within 1 week of culture. Three partly overlapping stages could be distinguished in the extracellular elastogenesis. First, small bundles of microfibrils appeared. These bundles then became associated with small conglomerates of a dense amorphous material. Finally, in the mature elastic fibers such material had fused into confluent amorphous areas within which no microfibrils were detected. This culture system with early and efficient production of extracellular matrix components may be a useful model for studies on aortic smooth muscle cells under various experimental conditions.

Fine structure of the Golgi complex during mitosis of cartilaginous cells in vitro

Chondrocytes were isolated enzymatically from guinea-pig epiphyses and grown in vitro. The fate of the Golgi complex during mitosis in relation to changes in the cytoplasmic microtubules was then studied by transmission electron microscopy. Interphase cells were observed to be polarized, with the Golgi complex occupying a well-defined juxtanuclear area of the cells cytoplasmic pole. During prophase the cytoplasmic microtublues were largely lost, the nucleus moved to the center of the cell and the Golgi complex dissolved into single dictyosomes spread diffusely throughout the cytoplasm. The distribution of other organelles also changed to a more random pattern. In telophase, i.e. after the completion of nuclear division, the mitotic spindle decomposed and cytoplasmic microtubules reappeared. Furthermore, the organization of the Golgi complex and other organelles returned to that characteristic of interphase cells. Previous studies on cells treated with colchicine have indicated that the polarized distribution of cell organelles is dependent on the presence of intact cytoplasmic microtubules. It is suggested that the disappearance of such tubules observed here to be coupled with the disorganization of cell interphase structure fulfills the double function of providing free tubulin units from which to build the mitotic spindle and ensuring an approximately equal distribution of dictyosomes and other organelles to the daughter cells during cytokinesis.

Fine structure of rabbit ear chondrocytes in vitro and after autotransplantation

SummaryChondrocytes were isolated enzymatically from rabbit ear cartilage, grown in vitro or as autotransplants for 1, 2 or 5 weeks and then examined by transmission electron microscopy. A confluent monolayer formed rapidly in vitro and the cells later grew in multiple overlapping layers, producing thick sheets of cartilaginous tissue. The cells retained a normal structure throughout the period of observation and, like the chondrocytes in intact cartilage, showed numerous microfilaments, an extensive granular endoplasmic reticulum and a prominent Golgi complex. Large amounts of intercellular matrix were laid down in vitro consisting of thin collagen fibrils, small rounded or polygonal granules believed to represent proteoglycans and patches and fibres of elastin.Chondrocytes in intramuscular autotransplants reconstituted an elastic cartilage. The exogenous origin of the cells in the transplants was verified by labeling of the lysosomes by exposure of the cells to colloidal thorium dioxide particles prior to injection. Structurally, the cells and the matrix of the transplants conformed to the above description. Accumulations of elastin-like material were sometimes observed in the Golgi vacuoles of the cells. Extracellularly, such conglomerates aggregated in connection with bundles of microfibrils, building up mature elastic fibres with a dense amorphous structure. The culture and transplant systems characterized here provide suitable experimental models for studies on development, growth and aging of elastic cartilage, including various aspects of the formation and turnover of elastic fibres and other macromolecular matrix components.

Electron microscopic studies on embryonic chick spinal ganglion cells:in vitro effects of antimicrotubular agents on the Golgi complex

SummarySpinal ganglia from 11 day chick embryos were incubated in media containing colchicine or vinblastine and subsequently examined by transmission electron microscopy. Both drugs caused a partial disappearance of cytoplasmic microtubules and a concomitant growth in the number of microfilaments in the neuroblasts. In ganglia treated with vinblastine for 4 h these effects were recorded morphometrically as a decrease of about 80% in the volume density of microtubules and a more than tenfold increase in the volume density of microfilaments. Furthermore, the cells displayed marked structural changes in the Golgi complex. The dictyosomes were mostly distinctly separated from each other and individual dictyosomes showed a decreased number of cisternae and an increased number of closely associated large vacuoles. The results are discussed with regard to the role of microtubules in the organization and function of the Golgi complex in nerve cells.

Electron microscopic studies on embryonic chick spinal ganglion cells: relationship between micro tubules and the Golgi complex

SummarySpinal ganglia from 11 day chick embryos were fixed immediately after removal, or after a short incubation in media with or without nerve growth factor (NGF), and were subsequently examined by transmission electron microscopy. Qualitatively, incubation did not effect the fine structure of the neuroblasts. Morphometrically, however, NGF was found to cause a marked increase in the amounts of microtubules and microfilaments in the perikarya of the cells with a threefold increase in the volume density of these organelles after 4 h. The cells displayed a prominent Golgi complex, mostly with dictyosomes organized in one continuous band or area. Individual dictyosomes consisted of a stack of 3–6 parallel cisternae associated with small vesicles and occasional larger vacuoles. Microtubules were observed in all parts of the cytoplasm but were particularly numerous within the Golgi area. They occurred both on the forming and maturing sides of the dictyosomes and in the latter site were closely associated with small vesicles and peripheral dilatations of the cisternae. These observations indicate a possible role of microtubules in the organization and function of the Golgi complex.

Heterotopic allotransplantation of isolated aortic cells

SummarySmooth muscle cells were enzymatically isolated from the tunica media of the aorta of 5-day-old rats. Following in vitro exposure to colloidal thorium dioxide particles the cells displayed numerous lysosomes filled with these particles. Intramuscular injection of isolated cells into the tongues of young rats of the same strain, resulted in the formation by both freshly isolated and thorium dioxide-labeled cells of a characteristic tissue similar to that found in the intact aortic wall. Thus, the transplants consisted of typical smooth muscle cells surrounded by an extracellular matrix consisting of a microfibrillar network, patches and fibers of elastin, collagen fibrils and small polygonal granules believed to represent proteoglycans. This system can be used for experimental studies on the production of extracellular matrix components and on other functions of arterial smooth muscle cells growing outside the vascular wall.The technical assistance of Ms. Karin Blomgren and the secretarial assistance of Ms. Inger Åhrén are gratefully acknowledged

Proteoglycans synthesized by fetal Guinea pig chondrocytes in culture

Short term cultures were carried out with chondrocytes and tissue fragments from fetal guinea pig epiphyseal cartilage. Proteoglycans were isolated from these cultures and their properties were compared with those of proteoglycans from adult hyaline cartilage. It was concluded that the proteoglycans synthesized in culture were essentially similar to those present in cartilage matrix in vivo. The authors therefore suggest that fetal guinea pig chondrocytes cultured in monolayer or as aggregates in suspension constitute a useful system for the study of synthesis and secretion of proteoglycans.

Elastin Formation in Heterotopic Transplants of Isolated Arterial Smooth Muscle Cells

Smooth muscle cells were isolated from the aorta of 5 day old rats by collagenase digestion and injected intramuscularly into animals of the same strain, where the cells reconstituted an elastic tissue with many similarities to that found in the media of the intact aortic wall. The transplants consisted of partly aligned smooth muscle cells surrounded by an extracellular matrix of microfibrils, elastic fibers, bundles of collagen fibrils, and small granules believed to represent proteoglycans. The production of extracellular matrix was much more efficient than in cultures of arterial smooth muscle cells. This cell transplantation system may be valuable in elucidating the mechanisms of normal growth and development of the arterial wall as well as the pathogenesis of various pathological processes.