Prostaglandin A2 and S uptake in connective tissue

Rats deficient in essential fatty acids (EFA) incorporated lesser amounts of radioactive sulfate into lung, kidney, spleen, heart, costal cartlidge, long bone and skull bone than did normal control animals. Administration of prostaglandin A₂ stimulated ³⁵S uptake by lung, kidney and aorta while ³⁵S levels in costal cartilage, tibial cap and long bone were strikingly reduced. Comments are presented suggesting that this metabolic mechanism may explain, in part, cartilage and bone resorption in areas of inflammation, such as arthritis, both rheumatoid arthritis and osteoarthritis.     

Prostaglandin A2 and 35S uptake in connective tissue

Rats deficient in essential fatty acids (EFA) incorporated lesser amounts of radioactive sulfate into lung, kidney, spleen, heart, costal cartilage, long bone and skull bone than did normal control animals. Administration of prostaglandin A2 stimulated 35S uptake by lung, kidney and aorta while 35S levels in costal cartilage, tibial cap and long bone were strikingly reduced. Comments are presented suggesting that this metabolic mechanism may explain, in part, cartilage and bone resorption in areas of inflammation, such as arthritis, both rheumatoid arthritis and osteoarthritis.     

Cartilage-derived factor (CDF) : II. Somatomedin-like action on cultured chondrocytes

Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [³⁵S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [³⁵S]sulfate incorporation but also [³H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [³H]uridine incorporation into rabbit chondrocytes and significantly enhanced [³H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit chondrocytes.     

Cartilage-derived factor (CDF) I. Stimulation of proteoglycan synthesis in rat and rabbit costal chondrocytes in culture

A protease-sensitive factor was extracted from fetal bovine cartilage with 1 M guanidine hydrochloride and partially purified by gel filtration on Bio-Gel A 0.5 m and CM-Sephadex column chromatography. This cartilage-derived factor (CDF) stimulated proteoglycan synthesis in rat and rabbit costal chondrocytes in culture, as shown by increased incorporation of ³⁵SO₄^?2, [³H]-glucosamine and [³H]serine into material precipitated with cetylpyridinium chloride. In addition, CDF stimulated the synthesis of sulfated glycosaminoglycans in a dose-dependent manner. These findings suggest that CDF is involved in the control of chondrogenesis.     

Phagocytic release of human leukocyte neutral proteases: Analysis of cartilage degradation products

Human polymorphonuclear leukocyte neutral proteases (HLNP) released during the process of phagocytosis of aggregated human gamma globulin were tested for their ability to degrade intact rabbit ear cartilage. Using ³⁵S-labeled cartilage as substrate, HLNP derived from 45 × 10⁷ cells released about 45% of the total incorporated ³⁵S. DE-52 chromatography of incubation supernatants revealed a single ³⁵S peak associated with minimal quantities of peptide or protein material as estimated by absorbance at OD_{230 + 280 nm}. Analytical ultracentrifugation gave a molecular weight of 51,800. Incubation of cartilage with excess α-chymotrypsin released ³⁵S-containing protein and peptide elements (M_r 79,400). Therefore, degradation, of the proteoglycans of intact cartilage by HLNP is more extensive than that noted with bovine pancreas α-chymotrypsin. The products of HLNP and α-chymotrypsin digestion of cartilage contained chondroitin sulfates A and/or C since both materials (after column chromatography) were sensitive to chrondroitinase ABC and AC digestion. Collagenolytic activity of HLNP is minimal compared to proteolytic activity as evidenced by the lack of hydroxyproline containing peptides released from cartilage during enzyme incubation. It is suggested that HNLP incubated with intact cartilage may serve as a relevant model in the search for new agents which could combat enzyme-mediated cartilage destruction.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

The N-Terminal Cleavage of Chondromodulin-I in Growth-Plate Cartilage at the Hypertrophic and Calcified Zones during Bone Development

Chondromodulin-I (ChM-I) is a 20–25 kDa anti-angiogenic glycoprotein in cartilage matrix. In the present study, we identified a novel 14-kDa species of ChM-I by immunoblotting, and purified it by immunoprecipitation with a newly raised monoclonal antibody against ChM-I. The N-terminal amino acid sequencing indicated that it was an N-terminal truncated form of ChM-I generated by the proteolytic cleavage at Asp³⁷-Asp³⁸. This 14-kDa ChM-I was shown by the modified Boyden chamber assay to have very little inhibitory activity on the VEGF-A-induced migration of vascular endothelial cells in contrast to the intact 20–25 kDa form of ChM-I (ID₅₀ = 8 nM). Immunohistochemistry suggested that 20–25 kDa ChM-I was exclusively localized in the avascular zones, i.e. the resting, proliferating, and prehypertrophic zones, of the cartilaginous molds of developing long bone, whereas the 14-kDa form of ChM-I was found in hypertrophic and calcified zones. Immunoblotting demonstrated that mature growth-plate chondrocytes isolated from rat costal cartilage actively secrete ChM-I almost exclusively as the intact 20–25 kDa form into the medium in primary culture. Taken together, our results suggest that intact 20–25 kDa ChM-I is stored as a component of extracellular matrix in the avascular cartilage zones, but it is inactivated by a single N-terminal proteolytic cleavage in the hypertrophic zone of growth-plate cartilage.     

Turnover of proteoglycans in guinea pig costal cartilage

The turnover in vivo of proteoglycans of guinea pig costal cartilage was investigated using Na₂³⁵SO₄ as precursor. Proteoglycans were extracted with guanidine · HCl, at both low and high ionic strength, and purified and fractionated by ultracentrifugation in CsCl gradients under associative and dissociative conditions. The results suggest that the sulfate is incorporated into macromolecules of at least two major metabolic pools with half-lives of about 3 days and about 60–70 days, respectively. Molecules with a fast turnover were enriched in the low ionic strength extracts and in fractions containing small, nonaggregated proteoglycans. No substantial evidence was found for a precursor-product relationship between different fractions.     

In vivo assay for somatic point mutations induced by genotoxic carcinogens: Incorporation of [35S]methionine into a rat liver cytochrome b5 normally lacking sulphur-containing amino acids

The trypsin fragments of rat liver microsomal cytochrome b₅ (Tb₅) lack both methionine (met) and cysteine (cys), i.e., the sulphur-containing amino acids. Tb₅ should therefore contain no ³⁵S-radioactivity after isolation from animals treated with [³⁵S]met or [³⁵S]cys. If, however, the nucleic acids coding for this polypeptide have been damaged by a genotoxic carcinogen, a miscoding could result in an incorporation of met or cys into the polypeptide so that Tb₅ could now be ³⁵S-radiolabelled. Two experiments are described, the first one where a toxic regimen of N-nitrosomorpholine (NNM) to rats resulted in a significant increase of ³⁵S-radioactivity in the Tb₅ of liver microsomes, and a second experiment with a non-toxic regimen of N,N-diethylnitrosamine (DENA), where no increase was observable.     

Keratan sulfate and related murine glycosylation can suppress murine cartilage damage in vitro and in vivo

Keratan sulfate (KS) proteoglycan side chains are abundant in the human cartilage matrix, but these chains have been said to be absent in murine skeletal tissues. We previously showed that KS suppresses cartilage damage and ameliorates inflammation in mice arthritis model. Because mice deficient of N-acetylglucosamine 6-O-sulfotransferase-1 (GlcNAc6ST-1) (KS biosynthesis enzyme) are now available, we decided to do further examinations.We examined, in culture, the difference between GlcNAc6ST-1^−/− and wild-type (WT) mice for interleukin (IL)-1α-induced glycosaminoglycan (GAG) release from the articular cartilage. Arthritis was induced by intravenous administration of an anti-type II collagen antibody cocktail and subsequent intraperitoneal injection of lipopolysaccharide. We examined the differences in arthritis severities in the two genotypes. After intraperitoneal KS administration in phosphate-buffered saline (PBS) or PBS alone, we evaluated the potential of KS in ameliorating arthritis and protecting against cartilage damage in deficient mice.GAG release induced by IL-1α in the explants, and severity of arthritis were greater in GlcNAc6ST-1^−/− mice than their WT littermates. Intraperitoneal KS administration effectively suppressed arthritis induction in GlcNAc6ST-1^−/− mice. Thus, GlcNAc6ST-1^−/− mice cartilage is more fragile than WT mice cartilage, and exogenous KS can suppress arthritis induction in GlcNAc6ST-1^−/− mice. Vestigial KS chain or altered glycosylation in articular cartilage in GlcNAc6ST-1^−/− mice may be protective against arthritis and associated cartilage damage as well as cartilage damage in culture. KS may offer therapeutic opportunities for chondroprotection and suppression of joint damage in inflammatory arthritis and may become a therapeutic agent for treating rheumatoid arthritis.     

N-acetylgalactosamine 4,6-bissulfate in rat urine II. Occurrence in rat cartilage and blood

The intraperitoneal injection of inorganic [³⁵S]sulfate to rat was followed by the rapid appearance in urine of a labeled compound which behaved as N-acetylgalactosamine 4,6-bissulfate on paper chromatography and paper electrophoresis and when treated with two sulfatases with a high degree of specificity toward the sulfate bonds at positions 4 and 6, respectively.Enzymatically-prepared N-acetylgalactosamine 4,6 [6-³⁵S]bissulfate was injected intravenously into rats. Of the injected dose, 90% was excreted unchanged in the urine during the subsequent 12 h, suggesting that the urinary N-acetylgalactosamine 4,6-bissulfate may derive from blood as renal filtrate.Examination of the rats injected with inorganic [³⁵S]sulfate revealed the presence of labeled N-acetylgalactosamine 4,6-bissulfate at significant levels in the blood and cartilage, but at much lower levels in the liver. The cartilage component was highest in its rate of ³⁵S uptake, suggesting that the blood component may derive at least in some part from the cartilage.Exposure of surviving cartilage slices to inorganic [³⁵S]sulfate, followed by extraction of the slices with hot 50% ethanol yielded a number of radioactive compounds, of which three were characterized as UDP-N-acetylgalactosamine-4,6-[³⁵S]bissulfate, N-acetylgalactosamine-1-phosphate 4,6-[³⁵S]bissulfate and N-acetylgalactosamine 4,6-[³⁵S]bissulfate. By subjecting the prelabeled tissue to chase incubation, it was possible to show that the UDP-N-acetylgalactosamine-4,6-bissulfate in the tissue disappeared with an approximate half-life of 10 min with a concomitant appearance in the medium of N-acetylgalactosamine 4,6-bissulfate and its 1-phosphate ester. These results suggest the occurrence in cartilage of an enzymatic system which is responsible for rapid turnover of UDP-N-acetylgalactosamine-4,6-bissulfate and possibly required for the rapid secretion of N-acetylgalactosamine 4,6-bissulfate into extracellular field.     

TAURINE IN DEVELOPING RAT BRAIN: SUBCELLULAR DISTRIBUTION AND ASSOCIATION WITH SYNAPTIC VESICLES OF [35S]TAURINE IN MATERNAL, FETAL AND NEONATAL RAT BRAIN

The concentration of taurine in the brain of the fetus in several species is higher than that found in the mature animal. In order to explore the functional significance of this, we have studied the subcellular distribution of taurine and [³⁵S]taurine in the brain of the mother, the fetus and the neonate after [³⁵S]taurine was administered to pregnant rats. In maternal brain, the distribution of taurine and of radioactivity (all of which was recovered from brain as taurine) in the subcellular fractions of maternal brain were essentially identical and were recovered primarily in two fractions (72% taurine, 71% [³⁵S]taurine was soluble, S₃; 16% and 17%, respectively, was in the crude mitochondrial and synaptosomal fraction, P₂). After further fractionation of P₂, most of the taurine and [³⁵S]taurine were in the cytoplasmic, O, and the synaptosomal, B, fractions. In the neonatal brain, shortly after birth there was a decrease in taurine and [³⁵S]taurine recovered in the supernatant fraction, S₃, accompanied by an increase in the percentage of taurine and [³⁵S]taurine recovered in the crude mitochondrial fraction. A small percentage of taurine and [³⁵S]taurine was consistently recovered in the synaptic vesicle fraction. Fractionation of the synaptic vesicles on a gel column separated the vesicle bound taurine completely from the free taurine: approx 1% of the taurine in the synaptic vesicle fraction was eluted with vesicles and could not be released by hypo-osmotic shock. The pattern of development in subcellular fractions of neonatal rat brain labelled with [³⁵S]taurine via intraperitoneal injections of the pregnant mother may be an indication of maturation or protection of putative taurinergic nerve endings.     

The effect of ascorbic acid oxidation on the incorporation of sulfate by slices of calf costal cartilage

A marked inhibition of the incorporation of S³⁵-sulfate by normal calf costal cartilage was produced by potassium ascorbate in the presence of catalytic amounts of cupric ions. The effect of the various components of the ascorbic acid oxidizing system (potassium ascorbate, cupric ions, cuprous ions, hydrogen peroxide, dehydroascorbic acid) was investigated. The results of experiments in which hydrogen peroxide, catalase, or sodium azide were used singly or in combination suggest that the inhibition produced by the ascorbic acid oxidizing system is due, to a considerable extent, to the production of hydrogen peroxide. Dehydroascorbic acid was also found to inhibit the incorporation of S³⁵-sulfate by cartilage slices. However, the gradual fall in pH which resulted from the addition of dehydroascorbic acid could account to a large extent for the inhibitory effect observed because the incorporation of S³⁵-sulfate by cartilage slices decreases sharply as the pH is lowered. The incorporation of S³⁵-sulfate by cartilage slices is inhibited also by increasing the concentration of phosphate.     

Effect of retinoic acid on chondrocyte glycosaminoglycan biosynthesis.

The effect of retinoic acid on glycosaminoglycan biosynthesis was investigated in rat costal cartilage chondrocytes in vitro. At levels of 10^?9 to 10^?8m retinoic acid, ³⁵SO₄ uptake into glycosaminoglycans was reduced 50%. At these low levels of retinoic acid there was no evidence of lysosomal enzyme release. The results are explained best in terms of modification of glycosaminoglycan synthesis, rather than accelerated degradation. Retinoic acid selectively modified the incorporation of ³⁵SO₄ or [¹⁴C]glucosamine into individual glycosaminoglycans fractions under the conditions studied. The relative incorporation of radiolabeled precursor into heparan sulfate (and/or) heparin increased three- to fourfold. The relative incorporation of radiolabeled precursor remained constant for chondroitin 6-sulfate, whereas incorporation into chondroitin 4-sulfate and chondroitin (and/or) hyaluronic acid decreased. Under the conditions studied, retinoic acid did not appear to be cytotoxic and did exhibit selective control over glycosaminoglycan biosynthesis. It is suggested that the decreased incorporation of ³⁵SO₄ into glycosaminoglycans at hypervitaminosis A levels of retinol may be accounted for by the presence of low levels of retinoic acid, a naturally occurring metabolite.     

Deficiency of fibroblast activation protein alpha ameliorates cartilage destruction in inflammatory destructive arthritis

IntroductionInflammatory destructive arthritis, like rheumatoid arthritis (RA), is characterized by invasion of synovial fibroblasts (SF) into the articular cartilage and erosion of the underlying bone, leading to progressive joint destruction. Because fibroblast activation protein alpha (FAP) has been associated with cell migration and cell invasiveness, we studied the function of FAP in joint destruction in RA.MethodsExpression of FAP in synovial tissues and fibroblasts from patients with osteoarthritis (OA) and RA as well as from wild-type and arthritic mice was evaluated by immunohistochemistry, fluorescence microscopy and polymerase chain reaction (PCR). Fibroblast adhesion and migration capacity was assessed using cartilage attachment assays and wound-healing assays, respectively. For in vivo studies, FAP-deficient mice were crossed into the human tumor necrosis factor transgenic mice (hTNFtg), which develop a chronic inflammatory arthritis. Beside clinical assessment, inflammation, cartilage damage, and bone erosion were evaluated by histomorphometric analyses.ResultsRA synovial tissues demonstrated high expression of FAP whereas in OA samples only marginal expression was detectable. Consistently, a higher expression was detected in arthritis SF compared to non-arthritis OA SF in vitro. FAP-deficiency in hTNFtg mice led to less cartilage degradation despite unaltered inflammation and bone erosion. Accordingly, FAP^−/− hTNFtg SF demonstrated a lower cartilage adhesion capacity compared to hTNFtg SF in vitro.ConclusionsThese data point to a so far unknown role of FAP in the attachment of SF to cartilage, promoting proteoglycan loss and subsequently cartilage degradation in chronic inflammatory arthritis.     

Uptake of l-[35S]Cystine by Isolated Rat Brain Capillaries

Adult rat brain capillaries were isolated by a simplified procedure and showed an enrichment of the marker enzyme, γ-glutamyltranspeptidase. The uptake of [³⁵S]cystine at 37°C by this preparation can be divided into two components, a sodium- and energy-dependent transport process for the free amino acid pool, with an apparent K_m of 36 μm , and a binding process, with an apparent K_m of 1.13 mm . Chemical analysis of the amino acid pool indicates that cystine is the major form of intracapillary ³⁵S. Cystine transport was not inhibited by lysine, but glycine, α-methylaminoisobutyric acid and β-2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid were inhibitory to a small extent.     

A “coupled” subchondral bone-articular cartilage tissue culture system for the study of cartilage proteoglycan metabolism

Summary Current evidence suggests that interactions between the subchondral bone and the articular cartilage of mammalian diarthrodial joints may occur through the action of bone-associated peptide factors. However, there is no suitable organ culture model for studying these interactions. This study defines a long-term tissue culture system where the articular cartilage is coupled to the adjacent subchondral bone obtained from the proximal ends of bovine metacarpals. Autoradiography done over 3 mo., by utilizing [³⁵S]SO₄ incorporation into cartilage proteoglycan (PG) and a procedure for cutting non-decalcified bone, demonstrated similar numbers of silver grains over chondrocytes in all cartilage zones, including the bone-cartilage interface. Newly synthesized PG (NSPG) from the cartilage of the “coupled” system over a 3-wk period was primarily of large hydrodynamic size (Kav of 0.34). Comparable bovine articular and nasal cartilage slice systems, incubated for short periods of time, yielded similar and somewhat larger NSPG, respectively. Labeled chondroitin sulphate PG accumulating in the medium of primary chondrocyte monolayer cultures, derived from the cartilage of the coupled system at 0, 1, 2, and 3 wk, revealed two polydisperse subpopulations (Kav of 0.30 to 0.38 and 0.51 to 0.68). We conclude that this coupled bone-cartilage system is viable for prolonged periods, is suitable for studies on the metabolism of articular cartilage PGs, and seems to have some advantages over the cultured articular cartilage slice system.     

Evidence that endogenous cyclic AMP does not modulate serum sulfation factor action on embryonic chicken cartilage

The role of cartilage cyclic AMP as a mediator or modulator of serum sulfation factor (SSF) action on embryonic chicken cartilage was assessed. Media with concentrations of rat serum (7.5%) sufficient to maximally stimulate chondromucoprotein synthesis as measured by ³⁵SO₄ incorporation did not change cartilage cyclic AMP levels. Theophylline (2.5mM) doubled cyclic AMP in cartilage incubated in media but had no effect on ³⁵SO₄ incorporation. In media containing 5% rat serum, theophylline at 0.5, 1.5 and 2.5mM caused a similar and significant rise in tissue cyclic AMP but only 2.5mM inhibited SSF stimulated ³⁵SO₄ incorporation. The data indicate that cartilage cyclic AMP neither mediates nor modulates SSF action on cartilage chondromucoprotein synthesis.     

Studies on the in vitro incubation procedure for [35]sulphate labelling of articular cartilage glycosaminoglycans

Articular cartilage from cow and calf femoral condyles was incubated in Tyrodes solution containing [³⁵S]sulphate for different periods up to 80 min. Glycosaminoglycans from the cartilage tissue and incubation medium were fractionated on Cetylpyridinium chloride and ECTEOLA cellulose microcolumns.The incorporation of [³⁵S]sulphate into all individual fractions of chondroitin sulphate and keratan sulphate was found to be linear from 20 to 80 min incubation time. As a rule the total specific activities of keratan sulphate and chondroitin sulphate were similar for both calves and cows.The proteoglycan material recovered from the medium amounted to about 1% of the tissue dry weight and was found to have a higher chondroitin sulphate: keratan sulphate ratio than the corresponding cartilage tissue for both calf and cow.The solubility profiles for the newly synthesised glycosaminoglycans, obtained from determination of the radioactivity in the individual fractions, were compared with those of glycosaminoglycans already present. These curves indicated that newly synthesised chondroitin sulphate had a higher average molecular size than that present in the tissue whereas the newly synthesised keratan sulphate had a smaller average molecular size. These newly synthesised components were also detected in the proteoglycans recovered from the incubation medium.     

Solubilization and anionic regulation of cerebral sedative/convulsant receptors labeled with [35S] tert-butylbicyclophosphorothionate (TBPS)

Binding activity for the cage convulsant [³⁵S]-$\text{tert}$-butylbicyclophosphorothionate, which appears to label a site closely associated with the chloride ionophore of the GABA_A/benzodiazepine receptor complex has been solubilized from rat cerebral cortex using the zwitterionic detergent CHAPS. Of several detergents screened, only CHAPS and CHAPSO were capable of solubilizing the binding activity with good recovery. The pharmacologic specificity of soluble [³⁵S]-$\text{tert}$-butylbicyclophosphorothionate binding is very similar to the membrane state. In both the membrane and soluble state, [³⁵S]-$\text{tert}$-butylbicyclophosphorothionate binding is enhanced by anions which support inhibitory post-synaptic potentials (“Eccles anions”), suggesting that [³⁵S]-$\text{t}$-butylbicyclophosphorothionate may label chloride channels thought to be involved in these potentials. Since this solubilization procedure also preserves GABA and benzodiazepine binding and their regulation by drugs such as barbiturates, purification and isolation of the macromolecular complex including chloride channel and GABA-benzodiazepine sites may be feasible.