Ulf Friberg

Electron microscopic demonstration of proteoglycans in guinea pig epiphyseal cartilage

Guinea pig epiphyseal cartilage was studied ultrastructurally after staining with ruthenium red or Alcian Blue. Extracellularly, the matrix granules were positively stained with both dyes, whereas no apparent staining of the collagen fibrils occurred. Intracellularly, a positive ruthenium red staining of the Golgi vacuole granules was observed. No Alcian Blue staining of the content of the vacuoles was detected. The fresh, untreated cartilage contained about 1.3% hexosamine on a dry weight basis. About three-fourths thereof represented chondroitin sulfate, the remaining part keratan sulfate and glycoproteins. After digestion of thin cartilage slices with hyaluronidase or chondroitinase ABC the chondroitin sulfate content was less than 50% of that in the buffer-incubated controls. Concomitantly, the matrix granules were reduced in size and number or completely absent. Papain digestion removed more than 90% of the chondroitin sulfate and all matrix granules. From these findings it was concluded that the matrix granules contain proteoglycans. Moreover, it seemed reasonable to assume that the Golgi vacuole granules, at least in part, represent intracellular proteoglycans. Signs of secretion of these granules by exocytosis were occasionally observed.

Ultrastructure and acid phosphatase activity of matrix vesicles and cytoplasmic dense bodies in the epiphyseal plate

The ultrastructure and acid phosphatase activity of matrix vesicles and cytoplasmic dense bodies in the epiphyseal plate of young guinea pigs were investigated. With respect to matrix structure, shape, and location within the intercellular space the vesicles could be divided into two distinct types. Both types were bounded by a triple-layered membrane and occurred in all zones of the cartilage. Matrix vesicles type I were rounded in shape and had a finely granular, electron-dense matrix. They were located in the longitudinal septa and were most numerous in the hypertrophying and calcifying zones where they often occurred in close vicinity of early mineral deposits. Vesicles type I were structurally indistinguishable from cytoplasmic dense bodies, and probably represent extruded dense bodies. The presence of acid phosphatase activity, as demonstrated by electron microscopic histochemistry, in cytoplasmic dense bodies and matrix vesicles type I strongly suggests that these structures are lysosomes. Matrix vesicles type II usually were markedly irregular in shape, contained a matrix which structurally was similar to the ground cytoplasm of the chondrocytes, and were most often found pericellularly.

Influence of colchicine and vinblastine on the Golgi complex and matrix deposition in chondrocyte aggregates. An ultrastructural study

Abstract Fetal guinea-pig epiphyseal chondrocytes were isolated enzymatically, aggregated, and the aggregates maintained in organ culture. As revealed by light and electron microscopy, the cultures produced a typical cartilaginous matrix, but no calcification occurred. Exposure of aggregating cells, or preformed aggregates, to colchicine or vinblastihe at 10 −5 M concentration led to disappearance of the microtubules, dissociation of the Golgi complex into single dictyosomes, and clustering of lysosomes. Thus, in treated cells the dictyosomes with accompanying vesicular structures were dispersed throughout the cytoplasm, whereas they were localized in a well-defined juxtanuclear region in control cells. The number and size of the cisternae forming a dictyosome were often reduced. Cells treated with vinblastine displayed macrotubules and an increased number of phagosomes. Both drugs reduced the deposition of intercellular matrix. In cells first exposed to either of the drugs for 2 or 5 days and then transferred to fresh medium for 3 or 6 days, the microtubules reappeared, the Golgi complex regained its normal appearance, and the amount of matrix increased. These findings are discussed in view of present concepts of the role of microtubules in cell secretion.

Influence of colchicine on the synthesis and secretion of proteoglycans and collagen by fetal guinea pig chondrocytes

Fetal guinea-pig epiphyseal chondrocytes were cultured in monolayers and as aggregates in the presence of antimicrotubular agents. Colchicine and vinblastine caused a dissociation of the Golgi complex, in addition to the disappearance of microtubules. Synthesis and secretion of proteoglycans and collagen were studied using radioactive precursors. Colchicine inhibited the synthesis of proteoglycans. The drug also inhibited secretion with an intracellular accumulation of these molecules. The proteoglycans secreted by the colchicine-treated cells had a smaller molecular size and contained a smaller proportion of aggregated molecules than proteoglycans in control cultures. However, there was no difference in the average size of the chondroitin sulfate side chains of the proteoglycan molecules. Nor was there any increase in the breakdown of proteoglycans in colchicine-treated cultures. Vinblastine was also found to inhibit synthesis and secretion of proteoglycans. Deuterium oxide also inhibited the synthesis of these molecules but stimulated their secretion into the medium. Colchicine caused an inhibition of both synthesis and secretion of collagen. It is suggested that the quantitative and qualitative effects of colchicine could be the result of disturbances in the Golgi complex, possibly in combination with a retarded translocation of secretory vacuoles. However, as the colchicine-treated chondrocytes were still able to continue a large part of their matrix biosynthesis with only moderate changes in the structure of the secreted molecules, it is probable that alternative pathways for the secretion of matrix molecules exist and/or the Golgi complex is able to retain a major part of its function despite the structural alterations.

Electron microscopic enzyme histochemical studies on the cellular genesis of matrix vesicles in the epiphyseal plate.

In order to study the cellular genesis of the matrix vesicles, the epiphyseal plate of the guinea pig was investigated with the electron microscope for the presence of acid phosphatase, ATPase, and alkaline phosphatase activity. Acid phosphatase activity was observed in cytoplasmic dense bodies in the chondrocytes and in matrix vesicles type I. Observations on the intracellular location of dense bodies containing acid phosphatase activity indicated that such bodies may be extruded in toto from the chondrocytes, thereby being transformed into matrix vesicles type I. Evidence thereof could be observed all over the epiphyseal plate but was most prominent in the zone of hypertrophy where it was often associated with an initial disintegration of the chondrocytes. The extrusion of dense bodies from the cells is suggested as representing a pathway for lysosomal enzyme secretion. After incubation for ATPase and alkaline phosphatase, electron-dense deposits occurred on the plasma membrane of the chondrocytes. Precipitate was also found on the limiting membrane of matrix vesicles type II, indicating that this membrane is most probably equivalent to the plasma membrane. The observations indicate that type II vesicles represent fragments, either of cellular projections, or of disintegrating cells.

Chemical and metabolic heterogeneity of chondroitin sulfate and keratan sulfate in guinea pig cartilage and nucleus pulposus

Abstract Chondroitin sulfate and keratan sulfate in guinea pig costal cartilage, nasal septum cartilage and nucleus pulposus were separated and fractionated by chromatography on CPC-cellulose, ECTEOLA-cellulose and Sephadex G-200 columns. Characterization of the chondroitin sulfates included determination of molecular weight and of the number, and position, of sulfate groups of the disaccharide units. Distinct chemical differences were found between the total fractions of chondroitin sulfate from the three tissues. Within the total fractions a marked heterogeneity was also apparent. The turnover of the two glycosaminoglycans was studied by using radioactive sulfate as precursor. The guinea pigs were killed at eight intervals, ranging from 30 min to 40 days after injection of the isotope. Total fractions of chondroitin sulfate from rib, nasal septum and nucleus pulposus showed half-lives of about 80, 40 and 30 days, respectively. In addition, the existence of a second metabolic component with a faster turnover (half-life about 3 days) was indicated in chondroitin sulfate from all three tissues. Similarly, keratan sulfate from the rib contained a ‘slow’ component with a half-life of about 90 days and a ‘fast’ component with a half-life of 4 days. For keratan sulfate from nucleus pulposus only a single component was demonstrable; it had a half-life of about 30 days. Within the total fractions of both chondroitin sulfate and keratan sulfate from the three tissues studied, a considerable degree of metabolic heterogeneity was apparent between, and within, subfractions of differing molecular weight.

In vitro influence of colchicine on the golgi complex in A- and B-cells of guinea pig pancreatic islets

Isolated guinea pig pancreatic islets were incubated (up to 4 hr) or cultured (up to 9 days) in the presence of colchicine. In control islets, the Golgi complex of the A-cells usually had a juxtanuclear location and was composed of a few dictyosomes. In the B-cells, the complex had a juxta- or perinuclear location and was considerably larger. Microtubules were present within and around the area occupied by the .Golgi complex. Exposure to colchicine caused, in addition to disappearance of the microtubules, marked changes in the Golgi complex. These occurred in a varying number of cells at 4 hr and in practically all cells at 24 hr. In both A- and B-cells, the dictyosomes became dispersed throughout the cytoplasm. The number and size of the cisternae were reduced in many of the cells. After transfer to a medium without colchicine, the Golgi complex regained its normal appearance. The above findings are interpreted as indicating that microtubules are responsible for the structural integrity of the Golgi complex. The changes in the dictyosomes are attributed to a postulated disruption of their functional relationship to the granular endoplasmic reticulum.

Electron microscopic and enzyme cytochemical studies on the guinea pig metaphysis with special reference to the lysosomal system of different cell types

SummaryA transmission electron microscopic study of demineralized, methaphyseal bone of the young guinea pig is presented. Special attention is paid to the lysosomal system of the different cell types. Visualization of acid phosphatase and aryl sulfatase activity was used to identify tissue components as belonging thereto. The distribution of alkaline phosphatase activity, a plasma membrane marker, was also examined.Osteoblasts were distinguished by a marked development of the granular endoplasmic reticulum and the Golgi complex. Perivascular cells type A, morphologically resembled the osteoblasts, and are believed to represent an early stage in the specialization of the latter. A few lysosomes were normally found in the osteoblasts; they were less common in the type A cells. In contrast to their regular occurrence in guinea pig epiphyseal cartilage, dense bodies of lysosomal nature (“type I vesicles”) were only rarely seen in the bone matrix. Structures analogous to the type II vesicles in cartilage were, however, normally present. Their membrane showed activity of alkaline phosphatase. Possible functions of lysosomes and matrix vesicles in osteogenesis are discussed.Perivascular cells type B and chondroclasts both contained a prominent Golgi complex and large numbers of free ribosomes, mitochondria and lysosomes. In the type B cells, inclusion material of varying appearance often occurred in the lysosomes and in endocytic vesicles. The chondroclasts sometimes presented a ruffled border, with associated vacuoles and lysosomes in the subjacent cytoplasm. It is suggested that both cell types participate in the resorption of the epiphyseal cartilage. Chondroclasts presumably arise by fusion of type B cells and/or monocytic precursors from the peripheral blood.The skilled technical assistance of Mrs. Eva Lundberg and the secretarial assistance of Mrs. Inger Åhrén are gratefully acknowledged.The authors are indebted to Dr. Stanislaw Moskalewski for constructive criticism of the manuscript.

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.

Cold and Metabolic Inhibitor Effects on Cytoplasmic Microtubules and the Golgi Complex in Cultured Rat Epiphyseal Chondrocytes

SummaryPrevious work has shown that exposure of cultured chondrocytes to colchicine leads to disappearance of microtubules and dispersion of the dictyosomes of the Golgi complex throughout the cytoplasm. Here, the effects of cold and metabolic inhibitors on cultured chondrocytes have been investigated in order to characterize further the relationship between these organelle systems. After incubation of cells for 24 h at 4° C most, but not all microtubules disappeared, indicating the existence of cold-resistant microtubules. Dictyosomes remained united in one area, until transfer of cultures to 37° C, when they dispersed throughout the cytoplasm in about one-third of the cells. In cells exposed simultaneously to cold and colchicine, microtubules disappeared completely, but spreading of dictyosomes occurred only in some cells and became generalized first upon warming. Application of the metabolic inhibitors sodium azide or sodium fluoride (10-2 M) or 2-deoxyglucose (5×10-2 M) together with sodium cyanide (10-2 M) inhibited microtubule removal by colchicine. Consequently, spreading of the Golgi complex was prevented. These findings support the concept of an important role of microtubules in the organization of the Golgi complex. Moreover, depolymerization of microtubules by colchicine appears to be an energy dependent process.