Heikki Saarni

A rapid method for separation and assay of radiolabeled mucopolysaccharides from cell culture medium.

Abstract A method is described for the separation and assay of radiolabeled glycosaminoglycuronans (GAGs) from cell culture medium. Following papain digestion and precipitation with cetylpyridinium chloride onto cellulose ester filter membrane, hyaluronic acid can be separated from sulfated GAGs by selective elution with HCl.

The decrease of hyaluronate synthesis by anti-inflammatory steroids in vitro.

The effect of anti‐inflammatory steroids (prednisolone and derivatives of hydrocortisone, dexamethasone and betamethasone) on the synthesis of hyaluronic acid and sulphated glycosaminoglycans in human skin fibroblast culture was studied. The concentrations of steroids varied between 1 × 10−10 M and 1 × 10−6 M. All tested steroids decreased the synthesis of hyaluronic acid to the same final level which was about 40–50% of the controls, but the concentrations required varied between different steroids. The relative inhibitory potencies of the steroids were calculated based on concentrations needed to decrease the synthesis of hyaluronate. When the inhibitory potency of hydrocortisone was calculated as one, the values of the other steroids were: prednisolone 5, hydrocortisone 17‐ butyrate 20, betamethasone alcohol 30, dexamethasone alcohol 38, betamethasone 17‐valerate 350–400, dexamethasone monosodium phosphate and betamethasone disodium phosphate over 400. Hydrocortisone sodium succinate was as potent an inhibitor of hyaluronate synthesis as hydrocortisone alcohol. None of the tested steroids affected the synthesis of sulphated glycosaminoglycans at these concentrations. The changes observed in the glycosaminoglycans in the medium were in accordance with the changes in the cell layer. The possible significance of hyaluronate synthesis inhibition by anti‐inflammatory steroids is discussed.

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

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

Synthesis of hyaluronic acid and collagen in skin fibroblasts cultures from patients with osteogenesis imperfecta

Collagen and hyaluronic acid syntheses were studied in skin fibroblast cultures from patients with osteogenesis imperfecta and age-matched controls by labeling the cultures either with [3H]proline and separating the collagenous proteins with DEAE- and CM-cellulose chromatographies, or double-labeling the cultures with [3H]glucosamine and [14C]glycine and separating radioactive hyaluronic acid from glycoproteins and sulphated proteoglycans by DEAE-cellulose chromatography. The activities of the cell layer hyaluronate synthesizing enzyme complex (hyaluronate synthetase) were also determined. The osteogenesis imperfecta cultures were classified into three variants on the basis of type III collagen synthesis. Type III collagen amounted to approx. 40--50% from total collagen in the first variety and approx. 25--30% in the second variety. No difference was noted in the ratio of type III collagen to total collagen in the third variety in comparison with control cultures. The radioactivities of 3H-labeled hyaluronic acid in DEAE-cellulose chromatograms were compared with those of the 14C-labeled proteins. The ratios ranged 9.2--17.3 in the cultures from the patients and 4.6--8.8 in the control cultures. Hyaluronate synthetase activities were 1.3--2.0-fold higher in the osteogenesis imperfecta cells than in their controls. Increased hyaluronic acid synthesis in skin fibroblasts correlated with the severity of the disease but not with the increase in type III collagen synthesis.

Effect of five anti-inflammatory steroids on collagen and glycoaminoglycan synthessis in vitro

The effect of five anti‐inflammatory corticosteroids, i. e. hydrocortisone, hydrocortisone 17‐butyrate, betamethasone 17‐valerate, nicocortonide acetate and nicocortonide, on the synthesis of hyaluronic acid, sulphated glycosaminoglycans and collagen by cultured skin fibroblasts was studied. As inhibitors of all these parameters the steroids could be arranged in order hydrocortisone < hydrocortisone 17‐butyrate < betamethasone 17‐valerate, nicocortonide acetate and nicocortonide. The corticosteroid concentrations required for inhibition of hyaluronic acid were very low as compared to those required for inhibition of sulphated glycosaminoglycan and collagen synthesis.

The effect of certain anti-inflammatory steroids on collagen synthesis in vitro

Abstract The effect of some anti-inflammatory steroids (prednisolone, and different derivatives of hydrocortisone, dexamethasone and betamethasone) on the synthesis and extra- and intracellular distribution of collagen was studied using chick embryo tendon cells. The concentrations of steroids varied between 1 × 10 −3 M and 1 × 10 −7 M. All steroids having an effect decreased the synthesis of collagen and the amount of labeled collagen decreased more in the medium than in the cells. Great variations in the inhibitory activity between different derivatives of steroids were noticed. Hydrocortisone was as inhibitory as hydrocortisone butyrate but both decreased collagen synthesis more than succinate or phosphate derivatives of hydrocortisone. Betamethasone-17-valerate was the most potent inhibitor among the steroids tested, whereas its alcohol and phosphate forms were ineffective. Dexamethasone and its phosphate derivative did not affect collagen synthesis in this system. The inhibitory potency of prednisolone on collagen synthesis was about the same as that of hydrocortisone. No steroid caused an accumulation of labeled collagen into cells and the decrease in the amount of collagen followed the decrease in total amount of labeled proteins. The results suggest that the inhibition of collagen synthesis by anti-inflammatory steroids is a consequence of a general inhibition of protein synthesis.

Cortisol decreases the synthesis of hyaluronic acid by human aortic smooth muscle cells in culture

The effect of cortisol on the synthesis of glycosaminoglycans (GAGs) was studied in cultured human aortic smooth muscle cells. Cortisol, at a level slightly exceeding the physiological concentration (10(-6) M), decreased the synthesis of hyaluronic acid (HA) by 50% but had no significant effect on the synthesis of sulphated GAGs. The ratio of HA to sulphated GAGs decreased by 47%. These effects were most marked in the fraction secreted into the culture medium. Cortisol neither affected the activity of the hyaluronic acid synthesizing enzyme complex in a cell-free system nor the molecular weight distribution of hyaluronic acid. We suggest that the atherogenity of cortisol and stress may be associated with their effect on the synthesis of HA by the smooth muscle cells of the arterial wall.

Cortisol effects on the glycosaminoglycan synthesis and molecular weight distribution in vitro

Abstract The effect of cortisol at different concentrations on the incorporation rate of [ 3 H]glucosamine and [ 35 S]sulphate into glycosaminoglycans (GAGs) in human fibroblast culture medium was studied. The mol. wt distribution of the synthesized GAGs was determined by Sepharose 2B chromatography. Two sensitivity levels of GAGs to cortisol were observed: at a low cortisol concentration ( 1 × 10 −7 M) only the hyaluronic acid synthesis decreased and no changes were observed in the synthesis of sulphated GAGs or glycoproteins. At a high steroid concentration (1 × 10 −3 M) both the synthesis of hyaluronic acid and sulphated GAGs drastically decreased. The mol. wt distribution of medium GAGs did not change at cortisol concentrations 1 × 10 −9 M-1 × 10 −5 M. The possible role of cortisol in the metabolism of hyaluronic acid in vivo is discussed.

Effects of cortisol on glycosaminoglycans synthesized by normal and rheumatoid synovial fibroblasts in vitro.

Cultivated rheumatoid synovial fibroblasts synthesize hyaluronic acid which has a changed molecular weight distribution in Sepharose 2B gel chromatography, when compared with normal synovial fibroblasts. Cortisol has no effect on the molecular weight distribution of either normal or rheumatoid hyaluronate. However, it does reduce the hyaluronic acid synthesis, by more than 50% in both cell types at low, near physiological plasma concentrations of the steroid.

Effect of hydrocortisone butyrate on collagen synthesis.

membranes have been rendered permeable for the fluorochrome by the hypotonic citrate solution (Krishnan, 1975). After filtration through nylon gauze a few drops of RNase (10 mg/ml) are added to remove extra-nuclear staining, and the cell suspension is ready for measurement. Fig. 2 shows a typical histogram obtained with this modified technique in comparison to that obtained using the method described previously. Advantages include the following: (1) The total time required for sample preparations is reduced from 3 h to 30 min. (2) Cell loss is minimal since no centrifugation steps are required. (3) Agglutination is minimal; even after x 100 amplification of the original signal, clumps of three cells can hardly be detected. (4) The variation coefficient (that is the width of the histogram) is reduced.