Variability of proteoglycan expression in the isolated rat glomerulus

The sulphation of proteoglycans in freshly isolated rat glomeruli was studied by biosynthetic labelling with [35S]sulphate. At least 75% of the observed sulphation requires de novo synthesis of core protein and proceeds at a constant rate over at least 40 h. Heparan and dermatan sulphate proteoglycans (HSPG and DSPG, respectively) are the two major species produced, with only minor amounts (less than 5%) of chondroitin sulphate labelled under these conditions. Several factors affect the population distribution of labelled material. When glomeruli were obtained from rats 6 weeks of age, HSPG accounted for 75 +/- 9% of tissue proteoglycan sulphated over 16 h. When older rats (12-14 weeks) were used, only 32 +/- 10% of label was associated with HSPG, DSPG accounting for the remainder. Production of HSPG is sulphate-dependent, increasing relative to DSPG with increasing sulphate, up to physiological concentrations. However, the net charge-density of sulphated material is conserved even at the lowest concentrations of sulphate. This may reflect the importance of electrostatic properties in the function of glomerular proteoglycans. The production of HSPG increases relative to DSPG with time following isolation and this effect is more dramatic in glomeruli from younger rats. However, reciprocal changes in production of HSPG and DSPG sustain a constant rate of sulphation. This phenomenon may arise from interdependency of the glomerular epithelial and mesangial cells with respect to regulation of proteoglycan synthesis.     

Renal glomerular proteoglycans. An investigation of their synthesis in vivo using a technique for fixation in situ.

Newly synthesized rat glomerular [35S]proteoglycans were labelled in vivo after injecting Na2[35S]SO4 intraperitoneally. At the end of the labelling period (7 h) the kidneys were perfused in situ with 0.01% (w/v) cetylpyridinium chloride. This fixed proteoglycans in the tissue and increased their recovery 2-3-fold during subsequent isolation of glomeruli from the renal cortex. The glomeruli were fractionated by a modified osmotic lysis and detergent extraction procedure [Meezan, Brendel, Hjelle & Carlson (1978) in The Biology and Chemistry of Basement Membranes (Kefalides, N.A., ed.), Academic Press, New York; Kanwar & Farquhar (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4493-4497] to obtain a basement membrane preparation. The proteoglycans released at each stage of the procedure were characterized using DEAE-Sephacel ion-exchange chromatography, chondroitinase ABC and HNO2 digestion and Sepharose CL-4B gel-permeation chromatography. About 85% of the [35S]proteoglycans synthesized were of the heparan sulphate variety, the remainder being chondroitin sulphate proteoglycans. Three sizes of heparan sulphate proteoglycans were identified. The largest (HS1, Kav. 0.47) accounts for 44% of the total extractable heparan sulphates. About one third of HS1 were extracted from the glomerular basement-membrane fraction with 8 M-urea and 4 M-guanidine hydrochloride but the remainder were released from the glomerulus during preparation of the fraction. The two smaller molecules (HS2, Kav. 0.56 and HS3, Kav. 0.68) accounted for 27% and 28% of the extractable heparan sulphate respectively and were not associated with the basement membrane fraction. HS1, HS2 and HS3 were also isolated from non-fixed glomeruli labelled in vivo but with much lower recovery. In glomeruli labelled in vitro, heparan sulphate accounted for only 35% of the proteoglycans, the remainder being of the chondroitin sulphate type. Proteoglycans similar to HS1, HS2 and HS3 were present in glomeruli labelled in vitro but, in addition, a large, highly charged heparan sulphate (HS1a) was extracted from the glomerular basement-membrane fraction of these glomeruli. It accounted for 6% of the total heparan sulphate.     

Isolation and characterization of proteoglycans synthesized by cultured mesangial cells

Rat mesangial cells selected by long-term culture of glomeruli exhibited a hill and valley appearance in the confluent state and were stained with antibodies against vimentin and desmin, suggesting that they are smooth muscle-like mesangial cells. The glycoconjugates produced by the cells were metabolically labeled with [35S]sulfate and [3H]glucosamine and extracted with 4 M guanidine HCl containing 0.5% Triton X-100. The radiolabeled glycoconjugates were separated on DEAE-Sephacel and compared with those synthesized by glomeruli labeled in the same conditions. Of the three major sulfated glycoconjugates, sulfated glycoprotein (17% of the total 35S-labeled macromolecules), heparan sulfate proteoglycan (35%), and chondroitin sulfate proteoglycan (30%) synthesized by glomeruli, the cultured mesangial cells synthesized mainly chondroitin sulfate proteoglycan (more than 90%). After purification by CsCl density-gradient centrifugation, the chondroitin sulfate proteoglycan from the cell layer was separated on Bio-Gel A-5m into three molecular species with estimated Mr values of 230,000, 150,000, and 40,000-10,000, whereas that released into the medium consisted of a single species with an Mr of 135,000. In the beta-elimination reaction, the former two larger proteoglycans released chondroitin sulfate chains with Mr of an apparent 30,000 and the latter from the medium released the glycosaminoglycan chains with an Mr of 36,000. The Mr of the smallest proteoglycan from the cell layer was not significantly changed after beta-elimination, indicating that this species had only a small peptide, if any. Analysis with chondroitinase AC-II and ABC demonstrated that all the chondroitin sulfates were copolymers consisting of glucuronosyl-N-acetylgalactosamine (65-74%) having sulfate groups at position 4 (53-57%) or positions 4 and 6 (10-14%) of hexosamine moieties and iduronosyl-N-acetylgalactosamine (21-26%) having sulfate groups at position 4 (17-23%) or positions 4 and 6 (about 3%) of hexosamine moieties; namely chondroitin sulfate H type. These characteristics of the chondroitin sulfate H proteoglycans synthesized by the cultured mesangial cells were very similar to those of the proteoglycans synthesized by glomeruli. Thus, we conclude that most, if not all, of the glomerular chondroitin sulfate proteoglycans are synthesized by mesangial cells. The cultured mesangial cells were also found to synthesize hyaluronic acid at a similar level to chondroitin sulfate proteoglycan. Based on the characteristics of this glycosaminoglycan, we discuss the possible role of hyaluronic acid produced by mesangial cells.     

Identification of the core proteins in proteoglycans synthesized by vascular endothelial cells.

Proteoglycans, metabolically labelled with [3H]leucine and 35SO4(2-), were isolated from the spent media and from guanidinium chloride extracts of cultured human umbilical-vein endothelial cells by using isopycnic density-gradient centrifugation, gel filtration and ion-exchange h.p.l.c. The major proteoglycan species were subjected to SDS/polyacrylamide-gel electrophoresis before and after enzymic degradation of the polysaccharide chains. The cell extract contained mainly a heparan sulphate proteoglycan that has a buoyant density of 1.31 g/ml and a protein core with apparent molecular mass 300 kDa. The latter was heterogeneous and migrated as one major and one minor band. After reduction, the apparent molecular mass of the major band increased to approx. 350 kDa, indicating the presence of intrachain disulphide bonds. The proteoglycan binds to octyl-Sepharose and its polysaccharide chains are extensively degraded by heparan sulphate lyase. The proteoglycans of the medium contained 90% of all the incorporated 35SO4(2-). Here the predominant heparan sulphate proteoglycan was similar to that of the cell extract, but was more heterogeneous and contained an additional core protein with apparent molecular mass 210 kDa. Furthermore, two different chondroitin sulphate proteoglycans were found: one 200 kDa species with a high buoyant density (approx. 1.45 g/ml) and one 100 kDa species with low buoyant density (approx. 1.3 g/ml). Both these proteoglycans have a core protein of molecular mass approx. 47 kDa.     

Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line

A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl- Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation.     

Inactivation of unbound rat liver glucocorticoid receptor by N-alkylmaleimides at sub-zero temperatures

Biosynthetically radiolabelled heparan sulphate proteoglycans have been isolated from the growth medium and the cell lysate of a human neuroblastoma cell line (CHP100). Chromatography on Sepharose CL-4B identified two heparan sulphate proteoglycans in the medium (Kav 0.220 and 0.389), whereas in the cell lysate the major proteoglycan species were more heterogenous and of a smaller overall molecular size (Kav 0.407) than the medium-derived counterparts. Chromatography on Sepharose CL-6B of free heparan sulphate glycosaminoglycan chains showed that the majority of cell-layer-derived material heparan sulphate 2, Kav = 0.509) was smaller than medium heparan sulphates (heparan sulphate 1 and heparan sulphate 2, Kav 0.230 and 0.317). Analysis of the patterns of polymer sulphation by nitrous acid treatment, gel chromatography and high-voltage electrophoresis established that in each heparan sulphate fraction there was on average 1.1 sulphate residues per disaccharide with an N:O sulphate ratio of 1.1. Heparan sulphate in the medium had a high proportion of di-O-sulphated disaccharides in regions of the chain with repeat disaccharide sequences of structure GlcA-GlcNSO3, whereas cell-associated material was enriched in di-O-sulphated tetrasaccharides of alternating sequences GlcA-GlcNAc-GlcA-GlcNSO3. The identification of several populations of heparan sulphate proteoglycans differing in molecular size and glycosaminoglycan fine structure may reflect the functional diversity of this family of macromolecules in the nervous system.     

Effects of 4-deoxy-L-threo-pentose, a novel carbohydrate, on neural cell proteoglycan synthesis and function.

A novel carbohydrate, 4-deoxy-L-threo-pentose (4-deoxyxylose), was synthesized by way of reductive dechlorination of a chlorodeoxy sugar. This carbohydrate, an analogue of xylose which is required for the initiation of glycosaminoglycan (GAG) synthesis, was used to explore the function of GAG side chains in neurite outgrowth on a laminin substrate. 4-Deoxyxylose inhibited the incorporation of 35SO4 into the GAGs of neuronal and astrocytic proteoglycans, with no effect being seen on the incorporation of [3H]glucosamine into proteoglycan. Direct analysis of the heparan sulphate fraction from such cells using nitrous acid digestion confirmed that the GAGs were undersulphated. No inhibition of either 35SO4 or [3H]glucosamine incorporation was observed in primary mouse hepatocytes exposed to 4-deoxyxylose. 4-Deoxyxylose produced a direct dose-dependent inhibition of neurite outgrowth by sensory neurons, and medium conditioned by neurons or astrocytes in the presence of 4-deoxyxylose displayed less laminin-complexed neurite-promoting activity than medium conditioned in its absence. These data suggest that 4-deoxyxylose inhibits neurite outgrowth by altering the sulphation of the GAGs of heparan sulphate proteoglycans.     

Bovine aorta contains at least two related forms of heparan sulphate proteoglycan

• 1.1. The proteoglycan peak from anion exchange chromatography of an extract of bovine aorta was digested with chondroitinase ABC. The residual heparan sulphate proteoglycans were further purified by chromatography on Sepharose CL4B and DEAE-Sephacel to yield two species, of high and low charge density.
• 2.2. Higher molecular weight material had a higher proportion of high charge density proteoglycan, while the lower molecular weight species had a higher proportion of low charge density heparan sulphate proteoglycan.
• 3.3. The two species shared epitopes as they both reacted with an antibody to heparan sulphate proteoglycan from bovine glomerular basement membrane.
• 4.4. On electron microscopy, both high and low charge density proteoglycans were visualized as ‘tadpole-like’ molecules, which showed a tendency to aggregate via their globular heads.
• 5.5. Bovine aortic smooth muscle cells were cultured in the presence of [³⁵S]sulphate and [³H]glucosamine. Proteoglycans were isolated from medium and cell layer extract by the methods outlined above.
• 6.6. The major HSPG species isolated from medium were significantly larger than those from cell layer and displayed substantial heterogeneity in both size of HS chain after papain digestion and size of protein core after heparitinase digestion. 7. The major cell layer species yielded two HS species of widely differing mol. wt after papain digestion, and a very small protein core after heparitinase digestion. Therefore cell layer-associated HSPGs show a good deal more homogeneity than those found in the medium.
• 7.8. Further ion-exchange chromatography after digestion with chondroitinase ABC revealed HSPG species of lower charge density, possibly derived from a hybrid chondroitin sulphate-dermatan sulphate proteoglycan (CS/DSPG) after removal of the CS/DS chains.

     

Binding of heparan sulphate and heparin to control and virus-transformed cells

A cloned embryonic mouse cell line contained specific cell-surface receptors for heparin and both the number and affinity appeared to be unchanged in a simian-virus-40-transformed subclone. In competitive binding assays heparan sulphate from the control clone was bound preferentially compared to that from the transformed subclone, indicating that the altered sulphation of heparan sulphate from transformed cells results in a lowered affinity for cell-surface receptors. Evidence was obtained suggesting that endogenous proteoglycans were not held at the cell surface by binding to these receptors alone. However the possibility that proteoglycans embedded in the plasma membrane may interact with the receptor has not been ruled out.     

Heparan sulfate proteoglycan from human and equine glomeruli and tubules

1. Proteoglycans were isolated from human and equine glomeruli or tubules by guanidine extraction and anion exchange chromatography. 2. These proteoglycan preparations contained about equal amounts of heparan sulfate and chondroitin sulfates. 3. During the preparation of glomerular or tubular basement membranes the main part of proteoglycans (greater than 50%) was extracted in the salt extract. Chondroitin sulfate proteoglycan was mainly found in the water and salt extracts of glomeruli and tubules, heparan sulfate proteoglycan in the deoxycholate extracts and the basement membranes. 4. The glomerular basement membrane (GBM) contains about 12% (human) or 20% (equine) of the proteoglycans of the total glomerulus. They consist of greater than 70% (equine) or 80% (human) of heparan sulfate. 5. Heparan sulfate proteoglycan was isolated from the proteoglycan preparations of human or equine glomeruli and tubules by additional treatment with nucleases and chondroitinase ABC followed by CsCl gradient centrifugation. 6. Protein accounts for about 40% (dry weight) of the heparan sulfate proteoglycans. Their amino acid composition is characterized by a high content of glycine, but 3-hydroxyproline, 4-hydroxyproline and hydroxylysine are lacking. 7. The biochemical characteristics of the heparan sulfate proteoglycan of human or equine glomeruli or tubules differ from that isolated from rat glomeruli by their higher protein content and their amino acid composition. The significance of these differences is discussed.     

Heterogeneity of cell-associated and secretory heparan sulphate proteoglycans produced by cultured human neuroblastoma cells

Biosynthetically radiolabelled heparan sulphate proteoglycans have been isolated from the growth medium and the cell lysate of a human neuroblastoma cell line (CHP100). Chromatography on Sepharose CL-4B identified two heparan sulphate proteoglycans in the medium (K_av 0.220 and 0.3890, whereas in the cell lysate the major proteoglycan species were more heterogenous and of a smaller overall molecular size (K_av 0.407) than the medium-derived counterparts. Chromatography on Sepharose CL-6B of free heparan sulphate glycosaminoglycan chains showed that the majority of cell-layer-derived material heparan sulphate 2, K_av=0.509) was smaller than medium heparan sulphates (heparan sulphate 1 and heparan sulphate 2, K_av 0.230 and 0.317). Analysis of the patterns of polymer sulphation by nitrous acid treatment, gel chromatography and high-voltage electrophoresis established that in each heparan sulphate fraction there was on average 1.1 sulphate residues per disaccharide with an N:O sulphate ratio of 1.1 Heparan sulphate in the medium had a high proportion of di-O-sulphated disaccharides in regions of the chain with repeat disaccharide sequences of structure GlcA-GlcNSO₃, whereas cell-associated material was enriched in di-O-sulphated tetrasaccharides of alternating sequences GlcA-GlcNAc-GlcA-GlcNSO₃. The identification of several populations of heparan sulphate proteoglycans differing in molecular size and glycosaminoglycan fine structure may reflect the functional diversity of this family of macromolecules in the nervous system.     

Production of proteoglycans by human lung fibroblasts (IMR-90) maintained in a low concentration of serum.

Maintenance of fibroblasts in 0.5% serum results in viable but non-proliferative cells that may be analogous to fibroblasts in vivo. The synthesis of proteoglycans by human embryo lung fibroblasts in Eagle's minimal essential medium with 0.5% newborn-bovine serum or with 10% serum has been compared. A similar amount of [35S]sulphate-labelled glycosaminoglycan per cell was secreted by fibroblasts in 10% or 0.5% serum. 35SO42-incorporation into sulphated glycosaminoglycans was enhanced in 0.5% serum when expressed per mg of cell protein, but [3H]glucosamine incorporation was decreased. The charge density of these glycosaminoglycans was not changed as determined by ion-exchange chromatography. It was concluded that decreased protein/ cell resulted in an apparent increase in 35S-labelled glycosaminoglycan synthesis/mg of cell protein, whereas decreased uptake of [3H]glucosamine resulted in a decrease in their glucosamine labelling. The proteoglycans secreted by fibroblasts in 0.5% serum were similar in glycosaminoglycan composition, chain length and buoyant density to the dermatan sulphate proteoglycan, which is the major secreted component of cells in 10% serum. Larger heparan sulphate and chondroitin sulphate proteoglycans, which comprise about 40% of the total secreted proteoglycans of cultures in 10% serum, were greatly diminished in the medium of cultures in 0.5% serum. The proteoglycan profile of medium from density-inhibited cultures in 10% serum resembles that of proliferating cultures, indicating that lack of proliferation was not responsible for the alteration. The dermatan sulphate proteoglycan, participating in extracellular matrix structure, may be the primary tissue product of lung fibroblasts in vivo.     

Effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix formation by bovine corneal endothelial cell cultures

The effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix (ECM) formation by cultured bovine corneal endothelial (BCE) cells was investigated. BCE cells actively proliferating on plastic dishes produced in the absence of xyloside an ECM containing various proteoglycans. Heparan sulfate was the main 35S-labeled glycosaminoglycan component (83%). Dermatan sulfate (14%) and chondroitin sulfate (3%) were also present. Exposure of actively proliferating BCE cells to xyloside totally inhibited synthesis of proteoglycans containing dermatan sulfate or chondroitin sulfate and caused an 86% inhibition of heparan sulfate proteoglycan synthesis. The heparan sulfate proteoglycans that were extracted from the ECM produced by BCE cells exposed to xyloside had a smaller size and a reduced charge density compared to their counterparts extracted from the ECM of cultures not exposed to xyloside. In contrast to the inhibitory effect of the xyloside on proteoglycan synthesis, exposure of actively proliferating BCE cells to xyloside stimulated synthesis of free chondroitin sulfate and heparan sulfate chains. All of the xyloside-initiated glycosaminoglycan chains were secreted into the culture medium. The proteoglycan-depleted matrices produced by BCE cells exposed to xyloside were used to study the effect of these matrices on proteoglycan synthesis by BCE cells. BCE cells growing on proteoglycan-depleted ECM showed a considerable increase in the rate of proteoglycan synthesis compared to BCE cells growing on normal ECM. Moreover, the pattern of glycosaminoglycan synthesis by BCE cells growing on proteoglycan-depleted ECM was changed to one which resembled that of BCE cells actively proliferating on plastic dishes. It is postulated that BCE cells are able to recognize when an ECM is depleted of proteoglycan and to respond to it by increasing their rate of proteoglycan synthesis and incorporation into the ECM.     

Proteoglycans synthesized in vitro by nude and normal mouse peritoneal macrophages

Peritoneal macrophages from nude mice were found to be functionally similar to 'activated' macrophages from normal mice. The objective of the present study was to characterize the proteoglycans synthesized and secreted in vitro by peritoneal macrophages isolated from nude and normal Balb/c mice and to investigate the relationship between macrophage 'activation' and changes in the proteoglycan patterns. Macrophages obtained by peritoneal lavage were seeded in Petri dishes. After 2 h incubation at 37 degrees C, the adherent cells (macrophages) were exposed to [35S]sulphate for the biosynthetic labelling of proteoglycans. After incubation, the cell and medium fractions were collected and analysed for proteoglycans and glycosaminoglycans. The glycosaminoglycans were identified and characterized by a combination of agarose gel electrophoresis and enzymatic degradation with specific mucopolysaccharidases. It was shown that 3/4 of the total 35S-labelled glycosaminoglycans were in the extracellular compartment after 24-48 h. The macrophages synthesized dermatan sulphate (68%), chondroitin sulphate (7%) and heparan sulphate (25%). Both cell and medium fractions of normal and nude mouse macrophages contained glycosaminoglycans with the same ratios, although the nude mouse macrophages synthesized 2-fold less glycosaminoglycans than the normal mouse macrophages. Lower levels of 35S-proteoglycans were also obtained from in vitro 'activated' macrophages, but the ratios of dermatan sulphate:chondroitin sulphate: heparan sulphate were altered in these cells as compared to the control. Furthermore, all the 35S-macromolecules found in the extracellular compartment of nude and normal control cells were of proteoglycan nature, in contrast to the medium fractions of 'activated' macrophages, which contain both intact proteoglycans and 'free' glycosaminoglycan chains. These results indicate that, at least as regards the proteoglycans and glycosaminoglycans, the nude mouse macrophages are not identical to the 'activated' macrophages from normal mice.     

Proteoglycan synthesis by normal and neoplastic human transitional epithelial cells

Metabolically 35S-labeled proteoglycans were isolated from cell-associated matrices and media of confluent cultures of human normal transitional epithelial cells and HCV-29T transitional carcinoma cells. On Sepharose CL-4B columns, the cell-associated proteoglycans synthesized from both cell types separated into three identical size classes, termed CI, CII, and CIII. Normal epithelial cell C-fractions eluted in a 22:34:45 proportion and contained 64%, 64%, and 72% heparan sulfate, whereas corresponding HCV-29T fractions eluted in a 29:11:60 proportion, and contained 91%, 77%, and 70% heparan sulfate, respectively. Medium proteoglycans from normal cells separated into two size classes in a proportion of 6:94 and were composed of 35% and 50% heparan sulfate. HCV-29T medium contained only one size class of proteoglycans consisting of 23% heparan sulfate. The remaining percentages were accounted for by chondroitin/dermatan sulfate. On isopycnic CsCl gradients, proteoglycan fractions from normal cells had buoyant densities that were higher than the corresponding fractions from HCV-29T cells. DEAE-Sephacel chromatography showed that cell and medium associated heparan sulfate from HCV-29T cells was consistently of lower charge density (undersulfated) than that from normal epithelial cells. In contrast, the chondroitin/dermatan sulfate of HCV-29T was of a charge density similar to that of normal cells. These as well as other structural and compositional differences in the proteoglycan may account, at least in part, for the altered behavioral traits of highly invasive carcinoma cells.     

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.     

Partial characterization of proteoglycans synthesized by human glomerular epithelial cells in culture

Confluent adult and fetal human glomerular epithelial cells were incubated for 24 h in the presence of [3H]-amino acids and [35S]sulfate. Two heparan-35SO4 proteoglycans were released into the culture medium. These 35S-labeled proteoglycans eluted as a single peak from anion exchange chromatographic columns, but were separable by gel filtration on Sepharose CL-6B columns. The larger heparan-35SO4 proteoglycan eluted with the column void volume and at a Kav of 0.26 from Sepharose CL-4B columns. The most abundant medium heparan-35SO4 proteoglycan was a high buoyant density proteoglycan similar in hydrodynamic size (Sepharose CL-6B Kav 0.23) to those previously described in glomerular basement membranes and isolated glomeruli. Heparan-35SO4 chains from both proteoglycans were 36 kDa. A smaller proportion of Sepharose CL-6B excluded dermatan-35SO4 proteoglycan was also synthesized by these cells. The predominant protein cores of both medium heparan-35SO4 proteoglycans were approximately 230 and 180 kDa. A hybrid chondroitin/dermatan-heparan-35SO4 proteoglycan with an 80-kDa protein core copurified with the smaller medium heparan-35SO4 proteoglycan. This 35S-labeled proteoglycan appeared as a diffuse, chondroitinase ABC sensitive 155-kDa fluorographic band in sodium dodecyl sulfate-polyacrylamide gels after the Sepharose CL-6B Kav 0.23 35S-labeled proteoglycan fraction was digested with heparitinase. The heparitinase generated heparan sulfate proteoglycan protein cores and the 155-kDa hybrid proteoglycan fragment had molecular weights similar to those previously identified in rat glomerular basement membrane and glomeruli using antibodies against a basement membrane tumor proteoglycan precursor (Klein et al. J. Cell Biol. 106, 963-970, 1988). Thus, human glomerular epithelial cells in culture are capable of synthesizing, processing, and releasing heparan sulfate proteoglycans which are similar to those synthesized in vivo and found in the glomerular basement membrane. These proteoglycans may belong to a family of related basement membrane proteoglycans.     

Inventory of human skin fibroblast proteoglycans. Identification of multiple heparan and chondroitin/dermatan sulphate proteoglycans.

Heparan sulphate and chondroitin/dermatan sulphate proteoglycans of human skin fibroblasts were isolated and separated after metabolic labelling for 48 h with 35SO4(2-) and/or [3H]leucine. The proteoglycans were obtained from the culture medium, from a detergent extract of the cells and from the remaining ''matrix'', and purified by using density-gradient centrifugation, gel and ion-exchange chromatography. The core proteins of the various proteoglycans were identified by electrophoresis in SDS after enzymic removal of the glycosaminoglycan side chains. Skin fibroblasts produce a number of heparan sulphate proteoglycans, with core proteins of apparent molecular masses 350, 250, 130, 90, 70, 45 and possibly 35 kDa. The major proteoglycan is that with the largest core, and it is principally located in the matrix. A novel proteoglycan with a 250 kDa core is almost entirely secreted or shed into the culture medium. Two exclusively cell-associated proteoglycans with 90 kDa core proteins, one with heparan sulphate and another novel one with chondroitin/dermatan sulphate, were also identified. The heparan sulphate proteoglycan with the 70 kDa core was found both in the cell layer and in the medium. In a previous study [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661] it was suggested that skin fibroblasts produce a proteoglycan form of the transferrin receptor. However, the core protein of the major heparan sulphate proteoglycan now purified does not resemble this receptor, nor does it bind transferrin. The principal secreted proteoglycans are the previously described large chondroitin sulphate proteoglycan (PG-L) and the small dermatan sulphate proteoglycans (PG-S1 and PG-S2).     

Metabolism of inorganic sulphate in the isolated perfused rat liver. Effect of sulphate concentration on the rate of sulphation by phenol sulphotransferase

1. The metabolism of inorganic [³⁵S]sulphate (Na₂³⁵SO₄) was studied in the isolated perfused rat liver at three initial concentrations of inorganic sulphate in the perfusion medium (0, 0.65 and 1.30mm), in relation to sulphation and glucuronidation of a phenolic drug, harmol (7-hydroxy-1-methyl-9H-pyrido[3,4-b]indole). 2. [³⁵S]Sulphate rapidly equilibrated with endogenous sulphate in the liver. It was excreted in bile and reached, at the lowest concentration in the perfusion medium, concentrations in bile that were much higher than those in the perfusion medium; at the higher sulphate concentrations, these concentrations were equal. The physiological concentration of inorganic sulphate in the liver, available for sulphation of drugs, is similar to the plasma concentration. 3. At zero initial inorganic sulphate in the perfusion medium, the rate of sulphation was very low and harmol was mainly glucuronidated. At 0.65mm-sulphate glucuronidation was much decreased and considerable sulphation took place, indicating efficient competition of conjugation by sulphation. At 1.30mm-sulphate the sulphation increased still further. 4. The results suggest that an important factor in sulphation is the relatively high K_m of synthesis of adenosine 3′-phosphate 5′-sulphatophosphate (the co-substrate of sulphation) for inorganic sulphate, which is of the order of the plasma concentration of inorganic sulphate. The steady-state adenosine 3′-phosphate 5′-sulphatophosphate concentration may determine the rate of sulphate conjugation of drugs in the rat in vivo.