Metabolism Of Sulfated Glycosaminoglycans Research Articles

Comparative35S-sulfate and3H-proline metabolism within the interdental septal bone and adjacent periodontal ligament

Tooth movements require rapid remodeling of the periodontal ligament (PDL) and adjacent alveolar bone. Our objective was to compare the regional metabolism of sulfated-glycosaminoglycans (sGAG) within the PDL and adjacent alveolar bone and compare it to the metabolism of collagenous proteins using radioautographic techniques. Rats were injected with either (3)H-proline or (35)S-sulfate and maxillae were removed at 1, 6, and 12 hr 1-7 days later. Silver grains were counted over the PDL and adjacent alveolar bone and the incorporation and removal rates for each radioisotope were determined. In general, net collagenous protein incorporation and removal were greatest within the distal and net sGAG incorporation and removal were greatest within the mesial compartments of the periodontium. The rate of removal of (3)H-proline was significantly greater within the distal alveolar bone surface than the adjacent PDL at all levels (P < 0.001). In contrast, the rate of removal of (35)S-sulfate was significantly greater in the PDL than within the adjacent mesial surface of the interdental septum at all levels (P < 0.001). The mesial surfaces of the interdental septum had a slower rate of removal of both isotopes than distal surfaces at all levels (P < 0.001). Our data suggest significant regional differences in the metabolism of (35)S-sulfate and (3)H-proline within the PDL and alveolar bone, which likely result from the characteristics of the forces produced by the adjacent teeth and may be a factor in the remodeling of the alveolar wall coincident to tooth movement.

Biosynthesis and metabolism of sulfated glycosaminoglycans during Drosophila melanogaster development.

We developed a simple methodology for labeling sulfated glycosaminoglycans (GAGs) in adult Drosophila melanogaster and studied some aspects of the biosynthesis and metabolism of these polymers during development. Adult D. melanogaster flies were fed with Na(2)(35)SO(4) for 72 h. During this period, (35)S-sulfate was incorporated into males and females and used to synthesize (35)S-sulfate-heparan sulfate (HS) and (35)S-sulfate-chondroitin sulfate (CS). The incorporation of (35)S-sulfate into HS was higher when compared to CS. In a pulse-chase experiment, we observed that (35)S-sulfate incorporated into adult female was recovered in embryos and used for the synthesis of new (35)S-sulfate-GAGs after 2 h of embryonic development. The synthesis of CS was higher than that of HS, indicating a change in the metabolism of these glycans from adult to embryonic and larval stages. Analysis of the CS in embryonic and larval tissues revealed the occurrence of nonsulfated and 4-sulfated disaccharide units in embryos, L1 and L2. In L3, in addition to these disaccharides, we also detected significant amount of 6-sulfated units that are reported here for the first time. Immunohistochemical analysis indicated that HS and CS were present in nonequivalent structures in adult and larval stages of the fly. Overall, these results indicate that (35)S-sulfate-precursors are transferred from adult to embryonic and larval tissues and used to assemble different morphological structures during development.

Static and dynamic compression modulate matrix metabolism in tissue engineered cartilage

Static and dynamic compression are known to modulate the metabolism of articular cartilage. The present study focused on determining the effects of compressive loading on the metabolism of sulfated glycosaminoglycans (S-GAG) and protein in tissue engineered cartilage constructs. Cartilage constructs were subjected to static or dynamic compression for 24 h and radiolabeled with 35 SO 4 and 3 H -proline to assess the total synthesis and percentage retention of S-GAG and total protein, respectively. Static compression at an amplitude of 50% suppressed the synthesis of both S-GAG and protein by 35% and 57%, respectively. Dynamic compression at an amplitude of 5% had stimulatory effects on synthesis that were dependent on the static offset compression amplitude (10% or 50%) and dynamic compression frequency (0.001 or 0.1 Hz). Thus, tissue engineered cartilage demonstrated the ability to respond to mechanical loading in a manner similar to that observed with articular cartilage. Mechanical loading may therefore potentially be used to modulate the growth of cartilaginous tissues in vitro, potentially facilitating the culture of functional cartilage tissues suitable for implantation.

Effects of Difloxacin on the Metabolism of Glycosaminoglycans and Collagen in Organ Cultures of Articular Cartilage

Effects of Difloxacin on the Metabolism of Glycosaminoglycans and Collagen in Organ Cultures of Articular Cartilage. Burkhardt, J. E., Hill, M. A., Lamar, C. H., Smith, G. N., Jr., and Carlton, W. W. (1993). Fundam. Appl. Toxicol. 20, 257-263.Fluoroquinolones, including difloxacin, are potent antibacterial compounds which, as a side effect, cause lesions in articular-epiphyseal cartilage complexes (AECC) of growing animals. To evaluate the effects of difloxacin on the structure of AECC and the metabolism of sulfated glycosaminoglycans (GAG) and collagen, explants of AECC were obtained from 18 healthy, 3-month-old Beagle dogs and cultured in medium which either had no difloxacin or had the drug at one of three concentrations (40, 80, or 160 μg/ml). Rates of synthesis of GAG and collagen were reduced by concentrations of difloxacin that were at or above 80 μg/ml. The rate of synthesis of total protein, however, was reduced only at the highest dose level. Catabolism of GAG and collagen was unaffected by the treatment. The principal ultrastructural changes in affected chondrocytes were distension of rough endoplasmic reticulum with electron-dense material that was probably protein, and vacuolation of cytoplasm. Structural changes were not observed in the extracellular matrix. It, therefore, appeared plausible that difloxacin affected chondrocytes by interfering with secretion of the matrix components, GAG and collagen.

Metabolism of glycosaminoglycans in CCl4-induced liver regeneration

The incorporation of [35S]-SO4 into glycosaminoglycans of liverin vivo and in in liver slices and into the glycosaminoglycans associated with the hepatic plasma membrane of rats at different periods after a heavy dose of CC14 have been studied. The incorporation of [35S]-SO4 into total glycosaminoglycans decreased to as low as 40% of the control at 24 h after the administration of CC14 and later on increased reaching a maximum on the 4th day. The amount of [35S]-SO4 incorporation into heparan sulphate was also reduced to about 40% of control at 12–24 h after the onset of injury and increased thereafter reaching a maximum on the 4th day. There was only a partial reduction in the synthesis of chondroitin sulphate in the early stage of injury and then it steadily increased reaching about 3 times the control level on 4–6 days. The [35S]-SO4-incorporation into dermatan sulphate, after a slight initial decrease remained at the control levels. On the 8th day after the CCl4-induced liver injury, the rate of [35S]-SO4-incorporation was almost equal to that in normal controls. The incorporation of [35S]-SO4 into hepatic plasma membrane glycosaminoglycans showed a similar change decreasing to about 35% of control at 24h followed by an increase, reaching normal levels on the 4th day after the administration of CC14. About 90% of the plasma membrane glycosaminoglycans was found to be heparan sulphate. The yield of plasma membrane from normal and CCl4-induced regenerating liver was found to be similar and therefore the results obtained were not due to difference in the yield of the membrane preparation. The data also indicate that there was no difference in the degree of sulphation. The significance of these changes in the metabolism of sulphated glycosaminoglycans particularly plasma membrane heparan sulphate in tissue regeneration has been discussed.

Effects of NSAIDs on the metabolism of sulphated glycosaminoglycans in healthy and (post) arthritic murine articular cartilage.

Several non-steroidal anti-inflammatory drugs (NSAIDs) were studied for their effects on normal and damaged murine articular cartilage, both in vitro and in vivo. In vitro, in the absence of serum, sodium salicylate caused significant suppression of 35S-glycosaminoglycan (GAG) synthesis, whereas tiaprofenic acid, piroxicam, prednisolone sodium phosphate and several other NSAIDs were without effect. Trypsin-mediated proteoglycan depletion did not change the susceptibility of the articular chondrocyte to these drugs. Similarly, no enhancement of drug effect was seen when arthritic cartilage was taken from an acutely inflamed joint, and prenisolone sodium phosphate even seemed to diminish inflammation-mediated suppression of 35S-GAG synthesis. The short term in vivo effects of some of the drugs were tested in mice with unilateral zymosan-induced arthritis. At day 1 after arthritis induction, in vivo 35S-GAG synthesis by the cartilage of the arthritic joint was decreased to 63%. Only sodium salicylate suppressed in vivo 35S-GAG synthesis in the healthy and arthritic joint to the same extent in both. At day 28, GAG synthesis in the postarthritic joint was enhanced to 160%. This type of cartilage appeared to be more susceptible to drug effects, since all drugs tested showed clear suppression of the augmented GAG production in vivo. Finally, in vivo drug effects were tested on normal and enhanced 35S-GAG degradation, the latter in the zymosan-induced arthritic joint. Both tiaprofenic acid and prednisolone sodium phosphate appeared to suppress degradation in healthy and, to some extent, in arthritic cartilage.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered expression of glycosaminoglycans in metastatic 13762NF rat mamma adenocarcinoma cells.

A difference in the expression and metabolism of sulfated glycosaminoglycans between rat mammary tumor cells derived from a primary tumor and those from its metastatic lesions has been observed. Cells from the primary tumor possessed about equal quantities of chondroitin sulfate and heparan sulfate on their cell surfaces but released fourfold more chondroitin sulfate than heparan sulfate into their medium. In contrast, cells from distal metastatic lesions expressed approximately 5 times more heparan sulfate than chondroitin sulfate in both medium and cell surface fractions. This was observed to be the result of differential synthesis of the glycosaminoglycans and not of major structural alterations of the individual glycosaminoglycans. The degree of sulfation and size of heparan sulfate were similar for all cells examined. However, chondroitin sulfate, observed to be only chondroitin 4-sulfate, from the metastases-derived cells had a smaller average molecular weight on gel filtration chromatography and showed a decreased quantity of sulfated disaccharides upon degradation with chondroitin ABC lyase compared to the primary tumor derived cells. Major qualitative or quantitative alterations were not observed for hyaluronic acid among the various 13762NF cells. The metabolism of newly synthesized sulfated glycosaminoglycans was also different between cells from primary tumor and metastases. Cells from the primary tumor continued to accumulate glycosaminoglycans in their medium over a 72-h period, while the accumulation of sulfated glycosaminoglycans in the medium of metastases-derived cells showed a plateau after 18-24 h. A pulse-chase kinetics study demonstrated that both heparan sulfate and chondroitin sulfate were degraded by the metastases-derived cells, whereas the primary tumor derived cells degraded only heparan sulfate and degraded it at a slower rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of sodium diclofenac on glycosaminoglycan metabolism in experimental osteoarthritis in rabbits.

The effect of diclofenac sodium (DS) on the metabolism of glycosaminoglycans (GAG) was studied in rabbits during the development of osteoarthritis of the knee induced by immobilization. Contents of hexosamines, uronic acid and sulphate-derived 35S-radioactivity in separated GAGs were determined. DS was given to 6 immobilized rabbits for 17 days at a dose of 1.5 mg/kg through a stomach tube. The controls consisted of 8 immobilized rabbits without medication and of 21 non-immobilized rabbits, 6 of which received DS for 17 days. DS did not inhibit in vivo metabolism of sulphated GAGs, according to measurements of [35S]-sulphate incorporation. Healthy rabbits given DS showed only marginal changes of GAG content in joint tissues. DS did not prevent the loss of GAG from the weight-bearing cartilages of the immobilized knees, but caused a further loss of chondroitin sulphates accompanied by an increased amount of hyaluronic acid in the tissues. DS prevented accumulation of the GAGs, which normally occurs during development of osteoarthritis in tissues of the tibial margin and in collateral ligaments. The findings indicate that the effect of DS on the GAG metabolism in connective tissues is somewhat different from that with other non-steroidal anti-inflammatory agents.

Regulation of heparan sulphate metabolism by adenosine 3':5'-cyclic monophosphate in hepatocytes in culture.

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E(1), and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of (35)SO(4) (2-) into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of (35)SO(4) (2-) or [(14)C]glucosamine into heparan sulphate was decreased to 40-50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse-chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by beta-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, (35)SO(4) (2-) incorporation was decreased to 5%. Addition of p-nitrophenyl beta-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl beta-d-xylosides. Incorporation of [(3)H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.

Metabolism of sulfated glycosaminoglycans in rat hepatocytes synthesis of heparan sulfate and distribution into cellular and extracellular pools

Primary cultures of rat hepatocytes grown in a serum-free medium supplemented with [ 35S]sulfate synthesize 35S-labelled glycosaminoglycans at an almost constant rate for 58 h. Approx. 57% of the newly synthesized 35S-labelled glycosaminoglycans remain within the hepatocytes, approx. 30% become associated with the cell-surface and only 13% are secreted into the medium. The amount of cell-surface-associated 35S-labelled glycosaminoglycans became constant within 36 h, whereas no equilibrium was reached in the intra- and extracellular pool. During a 24 h chase more than 50% of the intracellular and cell-surface-associated 35S-labelled glycosaminoglycans disappears, more than 80% of this material is degraded and radioactivity is recovered as inorganic sulfate. A minor part is released into the medium in a macromolecular form. Heparan sulfate accounts for more than 95% of the 35S-labelled glycosaminoglycans in each of the three pools. It is distinguished from heparan sulfates from other sources by the presence of unsubstituted glucosamine residues. In all three pools, heparan sulfate chains of mean molecular weights between 24 000 and 30 000 are part of an alkali labile proteoglycan. Intra- and extra-cellularly, however, part of the heparan sulfate appears to have little, if any, protein attached. Hepatocytes contain heparan sulfate-degrading endoglycosidase activity, which may contribute to the variation of molecular weights observed for the heparan sulfate.

Effect of lectins on the metabolism of sulfated glycosaminoglycans in cultured fibroblasts

A short exposure of human skin fibroblasts to Concanavallin A and wheat germ agglutinin led to an intra- and extracellular accumulation of sulfated glycosaminoglycans. The intracellular accumulation was caused by an impaired degradation of sulfated glycosaminoglycans. The increase of extracellular and cell surface associated 35S-labeled proteoglycans could be ascribed to a lectin-mediated inhibition of endocytosis of these polysaccharides. Results obtained with mono- and divalent Concanavalin A derivatives were in aggreement with the view that lectins inhibit endocytosis of sulfated proteoglycans by binding to the cell surface receptors specific for these polysaccharides. Proteoglycans secreted by fibroblasts formed predipitable complexes with Concanavalin A. Complex formation reduced markedly the uptake of the proteoglycan. All effects on glycosaminoglycan metabolism mediated by Concanavalin A and wheat germ agglutin could be prevented by methyl α-D-mannoside and N-acetylglucosamine, respectively.

Structure and metabolism of sulphated glycosaminoglycans in cultures of human fibroblasts. Structural characteristics of co-polymeric galactosaminoglycans in sequential extracts of fibroblasts during pulse-chase experiments

1. Human embryonic lung and skin fibroblasts were allowed to incorporate 32SO42- or 35SO42- and D-[1-3H]glucosamine. After removal of the medium the monolayer was subjected to sequential extractions by using EDTA, brief trypsin digestion, extraction with dithiothreitol ofllowed by freeze--thawing and extraction with trichloroacetic acid. The heparan sulphate and galactosaminoglycan contents of the various extracts were estimated after deaminative cleavage of the former component. Heparan sulphate was the major component of the trypsin digest, whereas galactosaminoglycans were the dominant component of other fractions. 2. Galactosaminoglycans of the various fractions were subjected to chemical (periodate oxidation/alkaline elimination) and enzymic (chondroitinase-AC and -ABC, as well as testicular hyaluronidase) degradations. Galactosaminoglycans from the insoluble cell fraction and the dithiothreitol extract contained larger amounts of L-iduronic acid than did those of other fractions. 3. Pulse-chase experiments were performed with and without replating of the cells at the start of the chase period. Radioactive glycans were isolated from the various extracts during the chase period. The half-lives of glycans of the insoluble cell fraction and the dithioreitol extract were shorter (5--8h) than were those of the trypsin digest and the EDTA extract (22h and 11h respectively). After replating of the cells in chase medium, radioactive cell-associated glycans were secreted from the cells and could be recovered in the trypsin digest, the EDTA extract and the medium. Furthermore, 35S/3H ratios of glycans from all these fractions decreased during the chase period. The following conclusions were reached. The insoluble cell fraction contains the synthesis pool and some structural material, whereas the soluble cell fraction is the storage and degradation pool. The dithiothreitol extract appears to contain the immediate precursors of secreted material. The trypsin-released glycans comprise structural components as well as material destined for pinocytosis or secretion into the medium. The EDTA extract is considered to consist of glycans en route to the medium. 4. The two presumptive precursor pools were preferentially depleted of L-iduronic acid-rich galactosaminoglycans during the chase. Glycans recovered from the trypsin digest, the EDTA extract and the medium during the chase contained larger amounts of periodate-resistant uronic acid residues (D-glucuronic acid and/or L-iduronic acid O-sulphate) than did their precursors. It is proposed that polymer-level modifications of secreted glycans are partly responsible for the results.

Metabolism of sulfated glycosaminoglycans in cultured endothelial cells and smooth muscle cells from bovine aorta

The glycosaminoglycan metabolism of cultured endothelial cells and of cells grown from the intima and from the media layer of bovine aorta thoracica was investigated in a comparative study. The following results were obtained: 1. 1. Endothelial cells have in common with intima and media cells the distribution of newly formed sulfated glycosaminoglycans into extracellular, pericellular and intracellular compartments. Endothelial cells, however, synthesize lower amounts of glycosaminoglycans and distribute them in a different ratio into the three pools. 2. 2. Though all the various cell lines synthesize chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, heparan sulfate and small amounts of keratan sulfate, endothelial cells exhibit a unique distribution pattern of sulfated glycosaminoglycans in each of the three compartments. Generally, a high proportion of heparan sulfate and chondroitin 6-sulfate and a very low dermatan sulfate content was detected. 3. 3. Heparan sulfate produced by endothelial cells has a higher N-sulfonate content when compared with that from other sources. The cell membrane-associated heparan sulfate, especially, exhibits some heparin-like features as judged by nitrous acid degradation and susceptibility towards heparitinase.

Sulfate Metabolism in Rat Calvaria Cultured Under Vitamin A Deficient Conditions

The metabolism of sulfated glycosaminoglycans (GAG) in bone has been found to be altered in vitamin A deficiency. A neonatal rat calvarium model system was used to determine if these changes are related to catabolic defects. Two day old rat pup calvaria were cultured in media containing serum from A- or A+ rats and radiolabeled sulfate or glucosamine. The incorporation of 35S-sulfate into the GAG fraction of calvaria cultured with A- serum for 48 hours was significantly increased compared to the values found in calvaria cultured with A+ serum (A-, 1,970 +/- 300; A+, 940 +/- 177; X +/- SD). The uptake of 35S-sulfate into the GAG fraction of calvaria cultured with A- serum showed continuous increase over 96 hours,whereas, 35S-sulfate uptake leveled off after 24 hours in the A+ group. There was also a significant increase in [14C]glucosamine uptake into the GAG fractions of calvaria cultured with A-serum (A-, 1,966 +/- 537; A+, 1,662 +/- 244; X +/- SD). To determine if the alteration in metabolism of sulfated GAG was in the biosynthetic or degradative pathways, a chase study was performed in which the calvaria were prelabeled with 35SO4. The rate of tissue loss of 35S-sulfate was lower in the total digests and the GAG fractions from calvaria cultured with A- serum than those cultured with A+ serum. Thus, the alteration in the metabolism of the sulfated GAG resulting from A- culture conditions seems to be defect in the degradative process.

Ammonium ion interferes with the lysosomal degradation of glycosaminoglycans in cultures of human glial cells

Summary Supplementation of human normal glial cells with NH 4 Cl led to marked effects on the metabolism of sulphated glycosaminoglycans (GAG). Sulphated GAG accumulated intracellularly in the presence of NH 4 Cl. Further, the degradation of sulphated GAG was partially inhibited. These phenomena were accompanied by an accumulation of lysosomes within the glial cells. The characteristics of NH 4 Cl-treated cells were thus similar to those displayed by cultured cells from patients with mucopolysaccharidosis.

Changes with age in the metabolism of sulfated glycosaminoglycans in guinea pig cardiac valves

1. 1|The formation and turn-over of sulfated glycosaminoglycans of the ground substance of cardiac valves was studied by autoradiography following the administration of S 33-sulfate in vivo to guinea pigs 10 days to 5 years of age. The experiments comprised a qualitative study involving all four heart valves in a group of 3-week-old animals, and a quantitative study on the aortic valve by age groups. The latter study included the use of testicular hyaluronidase for differentiation between chondroitin sulfates and dermatan sulfate. Sections from all the animals were examined for alterations with age in structural appearance and occurrence of metachromasia. 2. 2.|Moderate differences in structure and in amount and distribution of metachromasia were noted between growing and mature animals. There was no evidence, in 3- and 5-year-old guinea pigs, for degeneration with age such as has been observed in humans. 3. 3.|In the qualitative study, the cardiac valves showed a prominent incorporation of S 35, which equaled that of cartilage occurring in the aortic wall, trachea and bronchi. However, the turnover was more rapid in the valves than in cartilage. There was no significant difference between the four valves with regard to level of uptake, or turnover. 4. 4.|The metabolism of sulfated glycosaminoglycans is slowed with age, both in the rate of synthesis and in turnover time. 5. 5.|Digestion with testicular hyaluronidase indicated an increase with age in the proportion of dermatan sulfate in the ground substance.