Some aspects of the metabolism of sulfate-S35 and calcium-45 in the metaphyses of immature rats: influence of beta-estradiol benzoate

Weanling rats were given 2 mg. of 17-beta-estradiol benzoate at weekly intervals for 4 weeks. Twenty-four hours after each intraperitoneal injection of the estrogen 100 microc. of S(35)-sulfate or 11 microc. of Ca(45) was similarly injected. The animals were sacrificed 24 hours after the last dose of isotopes. An effect of estradiol benzoate on calcium metabolism was deduced from the observation that the concentration of calcium in some tissues of the treated rats was higher than the concentration in the tissues of untreated rats. Alkaline extracts of the distal metaphyses of femurs from the estradiol-treated and from control rats, given S(35)-sulfate, were shown by chromatography on an anion exchange resin to contain from 9 to 22 per cent of the S(35) as inorganic sulfate. From similar bone samples, 6 to 21 per cent of the S(35) was removed by decalcification with sodium versenate. Most of the remaining S(35) was associated with uronic acid and hexosamine; on paper chromatograms and paper electrophoretograms S(35) was shown to be part of material which migrated and was metachromatic in the same way as purified chondroitin sulfate. Autoradiograms of the proximal ends of tibiae from the animals given estradiol benzoate showed that both the S(35) and Ca(45) were deposited in the metaphyses in strata. The arrangement of the strata of S(35), however, was different from the arrangement of the strata of Ca(45). This difference in arrangement is interpreted as indicating that most of the S(35) in the metaphysis was derived from the chondroitin sulfate of the cartilage plate which the metaphysis had replaced.     

Histochemical and Autoradiographic Studies on the Effects of Aging on the Mucopolysaccharides of the Periosteum

An autoradiographic study was made using S³⁵-sulfate for the localization, distribution, and variation in the mucopolysaccharide content of the femoral periosteum of rats from birth to old age. The mucopolysaccharides were also studied histochemically, using toluidine blue O, Rinehart and Abu'l-Haj's colloidal iron method, and the periodic acid-Schiff reaction, before and after hyaluronidase treatment. Autoradiograms revealed the uptake of S³⁵ particularly in the vicinity of the preosseous zone and adjacent osteoblasts. This labelling was highest at the period of rapid bone growth. With increasing age, the S³⁵ uptake became progressively less. The preosseous zone showed γ-metachromatic staining at all ages after treatment with toluidine blue. Active osteoblasts were mostly orthochromatic, however, β-metachromasia was exhibited at a later age. Abundant amounts of intra- and extracellular mucopolysaccharides of both the acid and neutral type were demonstrated in the periosteum. S³⁵ uptake and γ-metachromasia show the presence of sulfated mucopolysaccharides, of which chondroitin sulfate predominates in the preosseous zone. Since S³⁵ uptake is high in active osteoblasts, the inability to demonstrate metachromasia in osteoblasts may indicate either that chondroitin sulfate is liberated as fast as it is being produced, or that it may be present within the cells in a precursor form not detectable by histochemical methods.     

THE EFFECTS OF FLUORIDE FEEDING ON THE ORGANIC MATRIX OF BONES AND TEETH OF PIGS AS OBSERVED BY AUTORADIOGRAPHY AFTER IN VITRO UPTAKE OF CA45 AND S35

Demineralized sections of fluorinated bones and teeth have been studied by autoradiography following in vitro uptake of Ca⁴⁵ or S³⁵O₄. The portions of tissue which do not become mineralized (cartilage, prebone, predentine, and precementum) show an increased Ca⁴⁵ uptake apparently related to an increase in chondroitin sulfate content in fluorosis. The tissues from the fluoride-fed animals show an increase of in vitro uptake of sulfur in the tissues which become mineralized (bone, dentine, cementum).     

CYTOCHEMISTRY OF PHOSPHATASES OF THE SARCOPLASMIC RETICULUM : I. Biochemical Studies

Observations were made on the behavior of chondrocytes grown under various conditions in vitro. The chondrocytes in 10-day embryonic chick vertebrae were grown as cultures of intact vertebrae, as pellets of chondrocytes liberated from their matrix, and as monodispersed cells plated out on plasma clots. Cartilage matrix was stained metachromatically with toluidine blue. Radioautographs were made of incorporated H³-thymidine, H³-proline, and S³⁵-sulfate to determine the extent of DNA synthesis, collagen synthesis, and chondroitin sulfate synthesis, respectively. Chondrocytes in intact vertebrae or in pellets are rounded and actively synthesizing chondroitin sulfate and collagen. There is little DNA synthesis by cells in either vertebrae or pellets. Chondrocytes grown as monodisperse cells rapidly cease synthesizing cytologically detectable chondroitin sulfate and are induced to synthesize DNA and divide. There is a change in the shape of these chondrocytes from a rounded to a more stellate condition which accompanies the shift in metabolic activity. Conversely, when the cells attain a certain cell density, they reacquire a rounded shape, cease dividing, and again synthesize chondroitin sulfate. Clusters of chondrocytes synthesize more chondroitin sulfate than isolated chondrocytes. It is concluded that most chondrocytes synthesizing chondroitin sulfate do not concurrently synthesize DNA. Interaction between associated chondrocytes is important in inducing and maintaining chondroitin sulfate synthesis in genetically determined chondrocytes. Failure of interaction between chondrocytes leads to DNA synthesis and cell multiplication.     

INTRACELLULAR SYNTHESIS OF CHONDROITIN SULFATE

In autoradiograms of slices of costal cartilage, incubated for 4 hours in a salt solution containing S³⁵-sulfate and then washed extensively and dehydrated, about 85 per cent of the radioactivity was assignable to the chondrocytes. From alkaline extracts of similarly prepared slices of cartilage, 64 to 83 per cent of the total sulfur-35 in the slices was isolated as chondroitin sulfate by chromatography on an anion-exchange resin. In view of the estimate that only about 15 per cent of the radioactivity was in the matrix, the isolation of 64 to 83 per cent of the total sulfur-35 as chondroitin sulfate is a strong argument that the chondrocytes are the loci in which chondroitin sulfate(s) is synthesized.     

AUTORADIOGRAPHIC STUDIES OF THE UTILIZATION OF S35-SULFATE BY THE CHICK EMBRYO

From studies of autoradiograms of various developmental stages of the chick embryo containing S³⁵ given us sulfate it was determined that as early as Stages 3+ and 4 there is a selective utilization or accumulation of sulfate by the various parts. The earliest accumulation site is the axial portion of the primitive streak and the floor of the groove. Later S³⁵ was found in the head process, Hensen's node, notochord, amniocardiac vesicle, wall of the omphalomesenteric vein, endocardium, subendocardial jelly, mesenchyme destined to become cartilage, basement membrane area of the gut, and a mucopolysaccharide layer formed on the free surface of the stomach. The early notochordal localizations of S³⁵ coincide with the region in which a thin ring of chondroitin sulfate is subsequently laid down. However, it is apparent that there is an intracellular accumulation of inorganic sulfate by the chondroitin-forming cells prior to the time they produce sufficient chondroitin sulfate to be demonstrable histochemically. It was interesting to note that the endocardium appears to concentrate sulfate that later apparently finds its way into the subendocardial jelly. The fact that those mesenchymal cells which later form chondroblasts begin to utilize sulfate selectively before histological differentiation is apparent was determined. In addition, the presence of sulfate-containing substances in the forming basement membrane of the gut would seem to indicate that sulfate is important in the histological differentiation of this membrane.     

TURNOVER OF S35-SULFATE IN THE MUCOSA OF THE GASTROINTESTINAL TRACT OF RATS AS SEEN IN AUTORADIOGRAMS

Segments of the gastrointestinal tract removed from rats after intervals of time following injection of S³⁵-sulfate were fixed in aqueous formalin and then washed in water. Contact and coated autoradiograms were prepared. The suggestion made by others that more of the labelled sulfate is fixed by the mucosa than by the underlying coats of the gastrointestinal tract is confirmed. In addition it was found that the isotope is fixed to a greater extent in the lower intestine than in the middle or upper portions of it. Coated autoradiograms revealed that 6 hours after administration of S³⁵-sulfate more of the label was present in the goblet cells lying deep in the crypts of the mucosa than in those adjacent to the intestinal lumen. By the 24th hour the concentration of the isotope was strikingly higher and more uniform from cell to cell. The mucus in the intestinal lumen was also highly radioactive. At the end of 48 hours very little of the sulfur-35 remained in the intestinal wall or could be made out in the mucus of the lumen: the autoradiographic reaction was faint and diffuse as contrasted with the punctiform and intense reaction given by the specimens removed at the end of shorter intervals of time.     

Studies on glycosaminoglycans of regenerating rabbit ear cartilage

Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitiated. Normal cartilage had a $\text{Δ}\text{Di-6S}\text{Δ}\text{Di-4S}$ ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.     

Age-dependent induction and repression of rat liver microsomal hydroxylase systems by estradiol

The activities of the hepatic microsomal 2α-, 2β-, 18- and 7α-hydroxylase systems active on 5α-[4-^14C] androstane-3α,17β-diol were studied in male and female rats which had been castrated and spayed at 14, 24, 35 and 45 days of age, treated for 5 days with 500 μg of estradiol benzoate per kg body weight and killed 6 days after gonadectomy. The hepatic microsomal 15β-hydroxylase enzyme system active on 5α-[1,2-³H] androstane-3α, 17β-diol 3,17-disulphate was also measured in these animals as well as in an additional group of animals that were gonadectomized at 56 days of age, treated with estradiol benzoate and killed at 62 days of age. The hydroxylase systems active on the free steroid substrate were all relatively unresponsive towards enstradiol in the developing rat, in contrast to the strong effects on hepatic hydroxylase activities previously noted following treatment of adult male rats with estradiol. On the other hand, the 15β-hydroxylase system active on disulphurylated 5α-androstane-3α, 17β-diol was inducible in both male and female rats of 41 and 51 days of age and in male rats of 61 days of age.     

The glycosaminoglycans of estrogen-induced medullary bone in Japanese quail

Glycosaminoglycans were isolated from the femurs of estrogen-treated male Japanese quail. During the 72 h after the injection of estrogen the incorporation of a 1-h pulse of H₂³⁵SO₄ into keratan sulfate increased more than 100-fold in a pattern corresponding to the production of the induced medullary bone. The rate of incorporation into chondroitin 4-sulfate, the only other glycosaminoglycan detected, remained constant throughout the same time period. The rate of incorporation of the 1-h pulse of sulfate into chondroitin 4-sulfate and keratan sulfate was the same at 48 h of estrogen treatment. When birds (48 h estrogen) were allowed to live 6 h after the injection of the isotope, chondroitin 4-sulfate accumulated 5-fold over that found for similar animals labeled for only 1 h. Keratan sulfate, into which the isotope was incorporated at the same rate as the chondroitin sulfate in this experiment, did not accumulate much more in 6 h of labeling than in 1 h of labeling. This suggests that the keratan sulfate turns over more rapidly than the chondroitin 4-sulfate in this tissue. Autoradiography showed that the chondroitin 4-sulfate was associated mainly with the marrow cells near the cortical bone and the keratan sulfate with the newly synthesized medullary bone. These results suggest that keratan sulfate is a specific marker for this secondary bone matrix.     

Aggregation of hydroxyapatite crystals

A system to study the aggregation of hydroxyapatite crystals was developed. The effect of several factors (Ca²⁺ × P_i product, Ca²⁺ /P_i ratio, pH, and various substances) were tested. Pb²⁺, Zn²⁺, Mg²⁺ and methyleneblue had only small effects; citrate inhibited aggregation. Pyrophosphate was a strong inhibitor and the diphosphonates disodium ethane-1-hydroxy-1,1-diphosphonate and disodium duchloromethylene diphosphonate were even more potent. The monophosphonate pentanemonophosphonate had no effect. Potent inhibition also occurred with glycosaminoglycans: heparin > hyaluronic acid > dermatan sulfate > chondroitin 4-sulfate > chondroitin 6-sulfate. Urine also showed high inhibitory activity. The inhibition of heparin but not that of hyaluronic acid, PP_i or urine was abolished by egg white lysozyme. The effects described might be relevant in the normal mineralization process as well as in the mechanisms leading to pathological calcification, such as urinary stone formation.     

Release of O-sulfate groups under mild acid hydrolysis conditions used for estimation of N-sulfate content

The treatment of chondroitin sulfate isolated from cultured B16 mouse melanoma cells with 0.04 M HCl at 100°C for 90 min released up to 45% of O-sulfate residues as free inorganic sulfate. In addition to the release of inorganic sulfate, extensive degradation of this polysaccharide as well as of cartilage chondroitin sulfate, pig rib cartilage proteoglycan, heparin and hyaluronic acid was also evident under these conditions. The above hydrolysis conditions are used for characterizing ³⁵S-labeled heparan sulfates synthesized by cultured cells and to calculate ratio of N- and O-sulfates in these molecules. Our results suggest that caution in necessary in interpreting the results of mild acid hydrolysis of glycosaminoglycans.     

Modulation of cAMP-dependent protein kinase by derivatives of chondroitin sulfate

Here, we report investigations about the direct effect of glycosaminoglycans, such as dermatan sulfate, chondroitin 4- and 6-sulfate upon cAMP-dependent protein kinase activity. The results indicate that glycosaminoglycans strongly influence the phosphorylation activity of this enzyme against histone type IIa and [Val⁶,Ala⁷]-kemptide. While chondroitin 4-sulfate and dermatan sulfate exhibit inhibitory effects, chondroitin 6-sulfate shows a stimulating effect. In addition, the chondroitin 6-sulfate is also able to reduce the chondroitin 4-sulfate and dermatan sulfate specific inhibition.     

Interactions between extracellular matrix components and proteoglycans released from monocytesin vitro

³⁵S-labelled chondroitin sulfate proteoglycans isolated from conditioned media of cultured human monocytes (day 1in vitro) and monocyte-derived macrophages (day 6in vitro) were chromatographed on columns of immobilized fibronectin and collagen, respectively. The elution profiles prior to and after alkali treatment were compared with those of standards chondroitin 4-sulfate and chondroitin sulfate E and heparin. The day 6³⁵S-proteoglycans have a higher sulfate density than the day 1 species, but this difference did not affect the elution profiles after chromatography on collagen-Sepharose, whereas the day 6 proteoglycans bound more firmly than the day 1 fraction to fibronectin-Sepharose. The elution patterns obtained for these distinct proteoglycans closely resembled those of heparin and oversulfated chondroitin sulfate E standards, and clearly demonstrated the importance of sulfate density both for the affinity to fibronectin and collagen. Neither day 1 nor day 6³⁵S-proteoglycans were found to interact with hyaluronate.Abbreviations used CSPG chondroitin sulfate proteoglycan - GAG glycosaminoglycan - CS chondroitin sulfate - CS-E chondroitin 4,6 disulfate - MDM monocyte-derived macrophages     

Mucopolysaccharide-polypeptide interactions: Effect of the position of the sulfate group

The differences in the interaction in solution of poly(l-lysine) with chondroitin 6-sulfate (chondroitin sulfate C) and with chondroitin 4-sulfate (chondroitin sulfate A) have been studied by circular dichroism spectroscopy. Both mucopolysaccharides force the poly(l-lysine) to adopt the α-helix in solution rather than the charged coil form expected at neutral pH. The observed spectra indicates that the polypeptide is at least 80% helical when the 6-sulfate form is present, but only about 20% α-helical in the presence of chondroitin 4-sulfate. Thus chondroitin66-sulfate has a stronger conformation directing effect on poly(l-lysine) than does the 4-sulfate, which is probably due to the different positions of the sulfate group on the polysaccharide c chain.     

The Compact and Biologically Relevant Structure of Inter-α-inhibitor Is Maintained by the Chondroitin Sulfate Chain and Divalent Cations

Inter-α-inhibitor is a proteoglycan of unique structure. The protein consists of three subunits, heavy chain 1, heavy chain 2, and bikunin covalently joined by a chondroitin sulfate chain originating at Ser-10 of bikunin. Inter-α-inhibitor interacts with an inflammation-associated protein, tumor necrosis factor-inducible gene 6 protein, in the extracellular matrix. This interaction leads to transfer of the heavy chains from the chondroitin sulfate of inter-α-inhibitor to hyaluronan and consequently to matrix stabilization. Divalent cations and heavy chain 2 are essential co-factors in this transfer reaction. In the present study, we have investigated how divalent cations in concert with the chondroitin sulfate chain influence the structure and stability of inter-α-inhibitor. The results showed that Mg²⁺ or Mn²⁺, but not Ca²⁺, induced a conformational change in inter-α-inhibitor as evidenced by a decrease in the Stokes radius and a bikunin chondroitin sulfate-dependent increase of the thermodynamic stability. This structure was shown to be essential for the ability of inter-α-inhibitor to participate in extracellular matrix stabilization. In addition, the data revealed that bikunin was positioned adjacent to both heavy chains and that the two heavy chains also were in close proximity. The chondroitin sulfate chain interacted with all protein components and inter-α-inhibitor dissociated when it was degraded. Conventional purification protocols result in the removal of the Mg²⁺ found in plasma and because divalent cations influence the conformation and affect function it is important to consider this when characterizing the biological activity of inter-α-inhibitor.     

Sulfated mucopolysaccharides of midgestation embryonic and extraembryonic tissues of the mouse

Incorporation of [³⁵S]sulfate into sulfated mucopolysaccharides has been characterized in midgestation mouse embryo, yolk sac, trophoblast, and decidua. Enzymatic analysis indicated that chondroitin sulfates contained approximately half of the label in embryo, trophoblast, and decidua, but less than 20% in yolk sac. While the labeled chondroitin sulfate fraction of trophoblast and decidua was mainly chondroitin-4-sulfate, only embryo contained a significant proportion of labeled chondroitin-6-sulfate. The relative incorporation into embryo chondroitin-6-sulfate was also substantially higher than that observed in four adult soft tissues. Labeled dermatan sulfate was absent from the embryo and yolk sac, but small amounts might have been synthesized by the placenta. Nitrous acid degradation studies revealed that essentially all the chondroitinase resistant MPS was N-sulfated, i.e., heparan sulfate and/or heparin. Electrophoretic profiles indicate that the bulk of the N-sulfated material resembles heparan sulfate rather than heparin. Electrophoretic heterogeneity and slow migration rates relative to standard markers suggest that the majority of labeled chondroitin sulfates may be undersulfated. The different mucopolysaccharide patterns in the various tissues may reflect their specialized properties and functions.     

The decreased synthesis of chondroitin sulfate-containing extracellular proteoglycans by SV40 transformed Balb/c 3T3 cells

Balb/c 3T3 cells synthesize 5–10 times more ³⁵SO₄^2?-labeled extracellular proteoglycan per cell than do Balb/c 3T3 cells transformed by SV40 (SV3T3). The extracellular ³⁵SO₄^2?-labeled proteoglycans of the Balb/c 3T3 and SV3T3 cells differ markedly in their acid mucopolysaccharide composition. Extracellular Balb/c 3T3 proteoglycans contain about 70–80% chondroitin sulfate, most of which is chondroitin 4-sulfate, and small amounts of heparan sulfate and/or heparin. On the other hand, extracellular SV3T3 proteoglycans contain 65–75% heparan sulfate and/or heparin and less than 15% chondroitin sulfate. Analysis of extracellular ³⁵SO₄^2?-labeled proteoglycan by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that Balb/c 3T3 alone synthesizes a class of proteoglycans capable of migrating in a 10% separating gel. This class of proteoglycans, designated as fraction C, accounts for up to 45% of the total extracellular Balb/c 3T3 ³⁵SO₄^2?-labeled proteoglycans and contains chondroitin sulfate exclusively. It is altogether absent in the extracellular SV3T3 proteoglycans. The absence of this and other classes of chondroitin sulfate-containing proteoglycans can account for the 5–10-fold decreased synthesis of ³⁵SO₄^2?-labeled proteoglycans by SV3T3 cells when compared to Balb/c 3T3 cells.     

Spondyloepiphyseal dysplasia, chondroitin sulfate type: a possible defect of PAPS--chondroitin sulfate sulfotransferase in humans

Four patients with an unusual form of spondyloepiphyseal dysplasia excreted in the urine undersulfated chondroitin 6-sulfate (Biochem. Med. $\text{7}$, 415–423, 1973). The sera of these patients show a low activity of PAPS — chondroitin sulfate sulfotransferase, while the undersulfated chondroitin sulfate present in their urine is a much better acceptor of ${}^{35}\text{SO}{}_4$ than standard chondroitin sulfate when they are incubated with [${}^{35}\text{S}$]PAPS and normal sulfotransferases. These results suggest that in these patients the skeletal lesions are secondary to a defect in the synthesis of chondroitin sulfate involving specifically the sulfotransferase activity.     

Effect ofin vitro differentiation on proteoglycan structure in cultured human monocytes

Parellel toin vitro differentiation of human monocytes into macrophage-like cells, the cells change their synthesis of glycosaminoglycans from chondroitin 4-sulfate to highly sulfated chondroitin sulfate, containing 4,6-disulfatedN-acetylgalactosamine units [Kolsetet al. (1983) Biochem J 210:661–67]. After exposure of monocyte cultures to [³⁵S]sulfate for 24h either from the onset of cultivation, prior to differentiation, or from day 4, after differentiation,³⁵S-macromolecules from medium and cell-layer were isolated and characterized. The cell-layer of day 5 cultures contained both proteoglycans and free polysaccharide chains, while the³⁵S-macromolecules present in the cell-layer of day 1 cultures and in medium of both monocytes and macrophage-like cells were almost exclusively of proteoglycan nature. Proteoglycans produced by macrophage-like cells were of larger size than the monocyte proteoglycans, most likely due to an increased polysaccharide chain length. These proteoglycans, in contrast to the monocyte-derived species, also showed affinity for fibronectin at physiological ionic strength.