Influences of sulfated glycosaminoglycans on biosynthesis of hyaluronic acid in rabbit knee synovial membrane

The effect of various sulfated glycosaminoglycans on glycoconjugates syntheses in synovial membranes of rabbit knee joints in culture was investigated by two different approaches. In the first approach, synovial membranes isolated from rabbit knee joints were cultured in the presence of sulfated glycosaminoglycans and [14C]glucosamine. In the second approach, solutions of sulfated glycosaminoglycans were injected into rabbit knee joints and synovial membranes isolated from the joints were cultured in the presence of [14C]glucosamine. The major part of [14C]glucosamine-labeled glycoconjugates associated with the synovial membranes and secreted into culture medium was hyaluronic acid. Of the natural glycosaminoglycans tested, dermatan sulfate gave the maximum stimulation of hyaluronic acid synthesis followed by chondroitin 4- and 6-sulfate. Heparin, heparan sulfate, keratan sulfate, keratan polysulfate, and hyaluronic acid had no significant effect. Of the chemically polysulfated glycosaminoglycans, GAGPS (a persulfated derivative of chondroitin sulfate) gave high stimulation but N-acetylchitosan 3,6-disulfate had no effect. The effect of sulfated glycosaminoglycans on hyaluronic acid synthesis was the same in both experimental approaches. The increase in the amount of secreted hyaluronic acid in culture medium paralleled that in synovial membranes. The results indicate that the galactosamine-containing sulfated glycosaminoglycans have a specific stimulatory effect on hyaluronic acid synthesis. A high degree of sulfation of the molecules appeared to potentiate the stimulatory effect.     

The effect of solubilized bone matrix fractions from different mammalian species on glycosaminoglycan synthesis by cultured fibroblasts

Human and bovine bone matrices were extracted with salt solutions of different composition and the extracts tested for stimulation of incorporation of radioactivity from [3H]glucosamine and [35S]SO4 into the hyaluronic acid and chondroitin sulfate of the cell pellet, the cell surface and the medium fractions of human synovial cells in culture. Stimulatory activity was extracted with a solution of 0.3 M EDTA in 2.5 M NaCl from bovine but not human bone. Subsequent extraction of the residues with 4 M guanidinium hydrochloride yielded activity from both matrices. A major stimulation of incorporation of radioactivity was observed in the cell surface fractions. Human synovial cells constitute a more sensitive assay system for the stimulatory activity than rabbit synovial cells.     

Growth-related production of proteoglycans and hyaluronic acid in synchronous arterial smooth muscle cells.

1. The growth-stimulating effect of serum on the proteoglycan and hyaluronic acid production in arterial smooth muscle cells was investigated, using cells synchronized by serum deprivation. 2. After stimulation, synthesis of [35S]sulfated proteoglycans and [14C]hyaluronic acid increased during G1 and G2 phases (about 2- and 5-fold, respectively, in the culture medium), in comparison with quiescent cells. 3. Neither the size, nor the charge, nor the relative proportions of [35S]glycosaminoglycans of the proteoglycans were modified. 4. However, when the cells were stimulated to divide, increased synthesis of large [14C]hyaluronic acid was observed concomitantly with the production of higher hydrodynamic size [35S]proteoglycans, which aggregated with hyaluronic acid (20%).     

Complexing of fibronectin glycosaminoglycans and collagen

Collagen-fibronectin complexes, formed by binding of fibronectin to gelatin or collagen insolubilized on Sepharose, were found to bind 20–40% of radioactivity in [³⁵S]heparin. Fibronectin attached directly to Sepharose also bound [³⁵S]heparin, while gelatin-Sepharose without fibronectin did not. Unlabeled heparin and highly sulfated heparan sulfate efficiently inhibited the binding of [³⁵S]heparin, hyaluronic acid and dermatan sulfate were slightly inhibitory, while chondroitin sulfates and heparan sulfate with a low sulfate content did not inhibit.The interaction of heparin with fibronectin bound to gelatin resulted in complexes which required higher concentrations of urea to dissociate than complexes of fibronectin and gelatin alone. Heparin as well as highly sulfated heparan sulfate and hyaluronic acid brought about agglutination of plastic beads coated with gelatin when fibronectin was present. Neither fibronectin nor glycosaminoglycans alone agglutinated the beads.It is proposed that the multiple interactions of fibronectin, collagen and glycosaminoglycans revealed in these assays could play a role in the deposition of these substances as an insoluble extracellular matrix. Alterations of the quality or quantity of any one of these components could have important effects on cell surface interactions, including the lack of cell surface fibronectin in malignant cells.     

Dibutyryl cyclic AMP affects hyaluronate synthesis and macromolecular organization in normal adult articular cartilage in vitro

When normal adult dog articular cartilage was cultured in the presence of dibutyryl cyclic AMP a higher proportion than normal of newly synthesized 35S-labeled glycosaminoglycans was released from the tissue into the culture medium, although their net synthesis was not affected. In conjunction with this release of sulfated glycosaminoglycans, 24 times more [3H]glucosamine-labeled hyaluronic acid was released from the cartilage into the medium, and net hyaluronate synthesis was enhanced 3-fold. Virtually all of the newly synthesized hyaluronic acid in the medium was associated with proteoglycans. The proteoglycans in the medium of the dibutyryl cyclic AMP treated cultures were normal in hydrodynamic size and interacted normally with hyaluronic acid to form large aggregates. These results suggest that the increase in hyaluronate synthesis caused by dibutyryl cyclic AMP mayt have destabilized the interaction of proteoglycans with the collagen meshwork of the cartilage. The changes seen in normal adult articular cartilage after incubation with dibutyryl cyclic AMP, therefore, are similar to those which are observed in cartilage of osteoarthritic joints.     

Heparin potentiates the action of plasma membrane-associated growth stimulatory activity

Plasma membranes prepared from mouse liver have been previously shown to contain growth stimulatory activity as determined with cultured mouse fibroblasts. This growth stimulatory activity, termed plasma membrane-associated growth stimulatory activity (PMGA), is highly mitogenic in the presence of platelet-poor plasma. We now demonstrate that the growth stimulatory action of PMGA is dramatically enhanced by the addition of heparin. The half-maximal effect of heparin was observed at 1-3 micrograms/ml. The synergistic effect was seen in two distinct assays; the stimulation of DNA synthesis in quiescent cells, and an increase of cell number over a 3-day culture period. Heparin, by itself, does not have any measurable influence on the growth of fibroblasts. The action of heparin is not unique to this glycosaminoglycan, as several other highly sulfated polysaccharides, including dextran sulfate, pentosan polysulfate, and fucoidan, also exhibited the highly synergistic effect. Among other glycosaminoglycans examined, chondroitin sulfate B and heparan sulfate had a small, but significant, effect on enhancing the growth stimulatory action of PMGA. Chondroitin sulfate A, chondroitin sulfate C, hyaluronic acid dextran, and poly-L-glutamic acid, however, had no detectable effect. Further experiments suggested that the effect of heparin is twofold, namely, both a potentiation of growth stimulatory activity and a protection of PMGA activity. The data presented here suggest that the association of various cell surface components, such as PMGA and specific proteoglycans, can modulate the growth potential of a cell.     

Evidence for an interaction between canine synovial cell proteoglycans and link proteins

Link proteins are glycoproteins which stabilize aggregates of proteoglycans and hyaluronic acid in cartilage. We recently identified link proteins in canine synovial cell cultures. We now find that link proteins and proteoglycans extracted from these cells under dissociative conditions sediment in the high-buoyant-density fractions of an associative cesium chloride density gradient, suggesting that link proteins interact with high-bouyant-density proteoglycans. In gradients containing [³⁵S]sulfate-labeled synovial cell extracts, 76% of the labeled sulfate and 54% of the uronic acid is found in the high-buoyant-density fractions. Under associative conditions, Sepharose 2B elution profiles of the crude synovial cell extract, synovial cell high-buoyant-density fractions, and culture medium indicate that synovial cell proteoglycans are present in monomeric form, rather than in aggregates. Synovial cell link proteins co-elute with the [³⁵S]sulfate-labeled material under the same conditions. These proteoglycans do not interact in vitro with exogenous hyaluronic acid. Dermatan sulfate, chondroitin sulfate and heparan sulfate are the major cell-associated sulfated glycosaminoglycans synthesized by cultured canine synovial cells, while hyaluronic acid is found in the culture medium. Although the proteoglycans synthesized by cultured synovial cells interact with link proteins, these data indicate that they do not interact with hyaluronic acid to form aggregates.     

Effects of hyaluronidase, trypsin, and EDTA on surface composition and topography during detachment of cells in culture

Cultured human embryo fibroblasts (HLM18) were labeled with [3H]glucosamine and Na35SO4, and then treated with testicular hyaluronidase, trypsin, or EDTA. Macromolecular material from the surface of these cells was characterized by DEAE-cellulose chromatography and cetylpyridinium chloride precipitation while the associated morphology of cell detachment was studied by phase contrast and scanning electron microscopy. Release of surface glycosaminoglycans by testicular hyaluronidase did not cause cell rounding or detachment. EDTA did not release cell-surface components, but caused cell contraction and detachment morphologically similar to that caused by trypsin. Large amounts of cell-surface glycoproteins and glycosaminoglycans were released by trypsin. From these observations it is concluded that hyaluronic acid is not a principal adhesive agent in the attachment of cells to a substrate. It is suggested that both EDTA and trypsin may have their primary effect upon the cytoskeleton.     

Stimulation by concanavalin A of cartilage-matrix proteoglycan synthesis in chondrocyte cultures

The effect of concanavalin A on proteoglycan synthesis by rabbit costal and articular chondrocytes was examined. Chondrocytes were seeded at low density and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of concanavalin A to the culture medium induced a morphologic alteration of the fibroblastic cells to spherical chondrocytes and increased by 3- to 4-fold incorporation of [35S]sulfate and [3H]glucosamine into large chondroitin sulfate proteoglycan that was characteristically found in cartilage. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, as chemical analyses showed a 4-fold increase in the accumulation of macromolecules containing hexuronic acid in concanavalin A-maintained cultures. Furthermore, the effect of concanavalin A on [35S]sulfate incorporation into proteoglycans was greater than that of various growth factors or hormones. However, concanavalin A had smaller effects on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant glycosaminoglycans. Since other lectins tested, such as wheat germ agglutinin, lentil lectin, and phytohemagglutinin, had little effect on [35S]sulfate incorporation into proteoglycans, the concanavalin A action on chondrocytes seems specific. Although concanavalin A decreased [3H]thymidine incorporation in chondrocytes, the stimulation of proteoglycan synthesis could be observed in chondrocytes exposed to the inhibitor of DNA synthesis, cytosine arabinoside. These results indicate that concanavalin A is a potent modulator of proteoglycan synthesis by chondrocytes.     

Incorporation of [3H]glucosamine into glycosaminoglycans in different cell culture conditions by rabbit aortic smooth muscle cells

This study sought to elucidate the optimal cell culture conditions for studies concerned with the incorporation of [³H]glucosamine into glycosaminoglycans by rabbit aortic smooth muscle cells. The incorporation of radioactivity into extracellular sulphated glycosaminoglycans was linear for at least 72 h and that into pericellular sulphated glycosaminoglycans for up to 24 h. The incorporation of radiolabel into hyaluronic acid was linear only up to 12 h. In the exponential growth phase the incorporation of [³H]glucosamine into sulphated glycosaminoglycans and hyaluronic acid proved to be less marked than in the stationary growth phase, but the highest values were nevertheless obtained immediately after trypsinisation. When studied in the stationary growth phase, cell density and incorporation of [³H]glucosamine were positively correlated in the case of hyaluronic acid, but in the case of sulphated glycosaminoglycans there was a negative correlation. The serum concentration of the incubation medium and the incorporation of radioactivity into hyaluronic acid were positively related. With sulphated glycosaminoglycans this was the case only after a 7-day preincubation in the different serum concentrations. when incorporation was studied without preincubation, the incorporation of radioactivity into sulphated glycosaminoglycans proved to be negatively associated with the serum concentration of the medium. The environmental pH of the cells was associated with the incorporation of radioactivity into hyaluronic acid and sulphated glycosaminoglycans in that between pH values 6.8 and 7.9 the incorporation of radioactivity increased when the pH of the medium was raised.     

Quantitative analysis of chondroitin sulphate retention by tannic acid during preparation of specimens for electron microscopy

Summary The ability of tannic acid to enhance binding of glycosaminoglycans to purified collagen was analysed in an in vitro system using amino sugar analysis on an amino acid analyser, transmission electron microscopy, and scanning electron microscopy. Collagen was purified by digestion with trypsin, papain, and hyaluronidase. Purified collagen was incubated with hyaluronic acid or with chondroitin sulphate glycosaminoglycan and then treated with tannic acid. Tannic acid was found to enhance retention during preparation for electron microscopy of either of the glycosaminoglycans onto collagen fibres. The ability of tannic acid to enhance binding of collagen and glycosaminoglycans might explain, at least in part, its structural reinforcement effect on resected synovial joint-apposing surfaces during preparation for scanning electron microscopy.     

Heparin inhibits skeletal muscle growth in vitro

Heparin or heparan sulfate proteoglycan (HeSPG), but not chondroitin sulfate or hyaluronic acid, exerts a pronounced inhibitory effect on muscle growth in vitro, as determined by total protein, myosin accumulation or synthesis, and [3H]thymidine incorporation studies. Primary muscle fibroblast culture growth is also inhibited by heparin but to a substantially lesser degree compared to muscle (30% and over 90% inhibition of growth, respectively). Heparin-induced inhibition of skeletal muscle growth is a consequence of its interaction with a growth factor(s) present in the media used to support myogenesis; heparin-Sepharose column absorbed horse serum can support muscle growth only in the presence of added heparin-binding growth factors like fibroblast growth factor (FGF) or chicken muscle growth factor (CMGF). Furthermore, heparin prevents the binding of iodinated FGF to the myoblast surface. We also show that the extent of muscle growth is a function of the relative amounts of heparin and FGF in culture. Finally, we provide evidence indicating that FGF can combine with endogenously occurring heparin-like components: immobilized FGF binds sodium-[35S]sulfate labeled components secreted in muscle culture conditioned medium, an interaction inhibited by anti-HeSPG antibodies or heparin, but not by other sulfated glycosaminoglycans. Since heparin binding growth factors not only stimulate myoblast proliferation but also actively inhibit the onset of muscle differentiation (G. Spitzz, D. Roman, and A. Strauss (1986). J. Biol. Chem. 261, 9483-9488), their interaction with naturally occurring heparin-like components may be an important physiological mechanism for modulating muscle growth and differentiation in development and regeneration.     

Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels.

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.     

Effect of vanadate on cartilage-matrix proteoglycan synthesis in rabbit costal chondrocyte cultures

The effect of vanadate on proteoglycan synthesis by cultured rabbit costal chondrocytes was examined. Rabbit chondrocytes were seeded at low densities and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of 4 microM vanadate to the culture medium induced a morphologic differentiation of the fibroblastic cells to spherical chondrocytes, and increased by two- to threefold incorporation of [35S]sulfate and [3H]glucosamine into large, chondroitin sulfate proteoglycans. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, in that chemical analyses showed increases in the accumulation of macromolecules containing hexuronic acid and hexosamine in vanadate-maintained cultures. However, vanadate had only a marginal effect on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant material. These results provide evidence that vanadate selectively stimulates the synthesis of proteoglycans characteristically found in cartilage by rabbit costal chondrocyte cultures.     

Structural features and some binding properties of proteoheparan sulfate enzymatically labeled by calf brain microsomes

Previous studies established that brain microsomes catalyze the transfer of [35S]sulfate from 3'-phosphoadenosine 5'-phospho[35S]sulfate to an O-linked oligosaccharide chain of a membrane glycoprotein and sulfamino groups of a membrane-associated proteoheparan sulfate (R. R. Miller and C. J. Waechter (1979) Arch. Biochem. Biophys. 198, 31-41). A large fraction of the proteoheparan [35S]sulfate can be released by treating the enzymatically labeled membranes from calf brain with 1 M NaCl. The salt-extracted 35S-labeled proteoglycan has been partially purified by a combination of ion-exchange and gel filtration chromatography. Based on chromatographic analyses, the 35S-labeled proteoglycan labeled in vitro is proposed to be a family of proteoheparan [35S]sulfates having an average molecular weight estimated to be 55,000. Variation in the length of the 35S-labeled polysaccharide chains partially accounts for the differences in molecular size of the proteoheparan [35S]sulfates. Binding studies reveal that the intact proteoheparan [35S]sulfates, as well as the free 35S-labeled polysaccharides released by mild alkali treatment, rapidly reassociate with calf brain membrane preparations. The association with calf brain membranes is saturable and reversible. Consistent with the binding being a specific interaction, only iduronic acid-containing glycosaminoglycans inhibit the association of the 35S-labeled proteoglycan with calf brain membranes and facilitate the disassociation. Neither the binding of the 35S-labeled proteoglycan to membranes nor the displacement was affected by hyaluronic acid, chondroitin 4-sulfate, or chondroitin 6-sulfate. The binding of the enzymatically labeled proteoheparan sulfate is reduced by preincubating membranes with either trypsin or chymotrypsin, but not with neuraminidase or phospholipase D. These results suggest that at least one class of proteoheparan sulfates could be specifically bound to one or more brain membrane proteins. The results also suggest a role for iduronosyl residues, and perhaps the stereochemical relationship of the carboxyl group to the O-sulfate moiety at C-2, in the recognition process.     

Glycosaminoglycans of arterial basement membrane-like material from cultured rabbit aortic myomedial cells

Arterial basement membrane-like material was prepared by a sonication-differential centrifugation technique from cultures of rabbit aortic myomedial cells after metabolic labelling with [35S]sulphate and [3H]glucosamine. Labelled glycosaminoglycans were obtained from isolated basement membrane-like material by proteinase digestion and gel filtration. Glycosaminoglycans were identified by a combination of Sephadex G-50 chromatography and sequential degradation with nitrous acid, Streptomyces hyaluronidase, testicular hyaluronidase and chondroitinase ABC. The data showed that heparan sulphate and chondroitin sulphate were the predominant glycosaminoglycans of myomedial basement membrane-like material. Heparan sulphate accounted for about 55% of [3H]glucosamine-labelled glycosaminoglycans. In addition small amounts of hyaluronic acid was present. Only trace amounts of dermatan sulphate was found. The glycosaminoglycans were analysed by DEAE-cellulose chromatography. Two major peaks were found in the chromatogram consistent with the predominance of heparan sulphate and chondroitin sulphate.     

Hyaluronic acid synthesis and secretion by rat liver fat storing cells (perisinusoidal lipocytes) in culture

The ability of rat liver fat storing cells to synthesize and to secrete hyaluronic acid was examined in monolayer cultures. The cells produce [3H] glucosamine-labeled hyaluronic acid, of which about 80% are secreted into the medium. The synthesis rate per cell (mg DNA) of labeled total glycosaminoglycans and hyaluronic acid in the medium increases significantly with culture time, but hyaluronic acid expressed as fraction of total glycosaminoglycans declines from about 0.70 in early cultures (up to the 4th day) down to 0.20 in advanced cultures. Cycloheximide increases and beta-D-xylopyranoside decreases significantly the fraction of hyaluronic acid in the medium, colchicine up to 5 microM was without effect. The synthesis of hyaluronic acid is a newly recognized function of this special type of sinusoidal liver cells. The results suggest that fat storing cells are likely to be a major source of hyaluronic acid in normal and probably also in injured liver.     

Stimulation of DNA synthesis in senescent human cells following incubation with plasma membranes

DNA synthesis and mitosis were increased in mitogen-stimulated senescent WI-38 cells following incubation with plasma membranes prepared from young or senescent WI-38 cells, A431 cells, 3T3 cells, or NR6 cells. The percentage of [3H]thymidine-labeled nuclei in senescent cultures was two- to fivefold greater than that seen in controls in which cells were incubated in the absence of membranes or in the presence of boiled membranes. The effect was trypsin sensitive, suggesting that a protein moiety is necessary for stimulation of DNA synthesis. As the culture age increased, basal levels of DNA synthesis, as well as maximal stimulation of DNA synthesis following incubation with plasma membranes, decreased. These observations are consistent with the hypothesis that different subpopulations exist in senescing cultures and suggest a complex pattern of inhibitory and stimulatory regulation of cell proliferation.     

Time and concentration dependence of the action of cortisol on fibroblasts in vitro

The effect of cortisol on cultured fibroblasts from human skin were studied. After 0–84-h preincubations in the presence of cortisol the cells were labeled for 12 h with [³H]thymidine, [³H]proline or [³H]glucosamine and the radioactivity incorporated into DNA, collagen, total proteins, hyaluronic acid and sulphated glycosaminoglycans was determined.Cortisol (1 · 10^?5 M) caused a rapid, progressive decrease in the synthesis of hyaluronic acid when compared to the controls. Similarly, it decreased the synthesis of sulphated glycosaminoglycans and DNA, but this was seen first after 12- and 24-h preincubations, respectively. The synthesis of collagen and other proteins was significantly increased when the preincubation time was 0–24 h. This stimulation, however, turned to inhibition when an 84-h preincubation was used. It was found that 1 · 10^?7 M cortisol was the lowest concentration which caused the early inhibition in hyaluronate synthesis, while even 1 · 10^?8 M was sufficient after an 84-h preincubation. The syntheses of sulphated glucosaminoglycans and DNA were significantly inhibited by 1 · 10^?8 and 1 · 10^?7 M cortisol, after an 54-h preincubation, respectively. Thus, the studies of cortisol effects on fibroblast functions may result in quite variable conclusions unless the time sequence and the steroid concentration effects are taken into account.     

Effect of thyrotropin on glycosaminoglycans synthesized by primocultured thyroid cells

The synthesis of glycosaminoglycans (GAGs) was investigated in porcine thyroid cells under the influence or not of thyrotropin. After labelling with [³H] glucosamine and [³⁵S] $\text{SO}{}_4{}^{\mathrm{2?}}$, enriched GAG-fractions prepared from culture media, cells, and eventually substrate adhering materials, were analyzed by cellulose acetate electrophoresis combined with specific degradations. They comprised heparan sulfate and hyaluronic acid together with an unknown sulfated component labile to endo-β-galactosidase. Whereas global labellings of newly made GAGs were not significantly modified by thyrotropin, we reproducibly observed with the hormone a substantial increase in the proportion of hyaluronic acid [³H] label and, when cells organized into follicles, of the proportion of cell-associated [³H] GAGs. This system thus offers an interesting model to study how the responsiveness to an hormone and the reorganization that follows might implicate specific glycoconjugates.