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Acid mucopolysaccharides in fibroblast cultures
Exp. Path., Bd. 12, S. 159-162 (1976) Friedrich-Schiller-University J ena, Institute of Pathology (Head: Prof. Dr. sc. med. F. BOLCK)
Acid mucopolysaccharides in fibroblast cultures 2. 3S S-sulfate incorporation kinetics in dependence on pH-value and cell density By P. D. KITTLICK, G. NEUPERT, K. J. STILLER and F. BOLCK With 2 figures (Received July 15, 1975) Key-words: acid mucopolysaccha,rides (MPS); 3sS-su lfate incorpora,tion; cell culture; cell density; pH-value; chondroitin sulfate; heparan sulfate; dermatan sulfate; lactate concentration; liquid scintillation; fibroblasts
Summary Cultures of embryonic rat fibroblasts were incubated with 3sS-sulfate at pH 6.6 and 7.4 (Eagle ba,saJ medium plus HEPES buffer) for 12 to 48 hours. The acid mucopolysaccharides wereisola,ted and fra,ctionated after the method of SVEJCAR and ROBERTSON. Sulfate incorporation was determined by liquid scintillation counting. Results: 1. In proliferating cultures linear rise of the specific activity of MPS (heparan sulfa,te, chondroitin-G-sulfate and dermatan sulfate) was observed for 48 hours. In stationary cultures balance of synthesis and breakdown occurs beginning with the 24th hour. The results apply to pH 6.G and 7.4. 2. The specific activity of dermatan sulfate is preponderating over that of chondroitin-G-sulfate. It is enha,nced by growing cell density. 3. Lactate addition to the culture medium at pH G.G a,nd 7.4 results in qualitatively similar effects. Abbreviations: Ch-6-S: chondroitin-G-slllfate CPC: cetylpyridinium chloride DS: dermata,n sulfate HA: hyaluronic a,cid HS: hepa.ran sulfa.te MPS: a,cid mucopolysaccharides, glycosaminoglycans S.A.: specific activity (cpm/fig MPS) The actual content of MPS in tissues and cell cultures represents a balance of de novo-synthesis and breakdown. In our previous paper we reported on the influence of pH value, lactate concentration and cell density on the content and distribution pattern of MPS in secondary cultures of embryonic rat fibroblasts (KITTLICK et a!. 1976, 1). In this as well as in two future papers we will describe the results of our simultaneous studies on 3sS-sulfate incorporation into HS, Ch-6-S and DS. Our findings give hints to what an extent de novo-synthesis and breakdown processes take part in the modification of the MPS-distribution pattern. The present study deals with the incorporation kinetics of 3sS-sulfate into the MPS-fractions HS, Ch-6-S and DS at pH 7.4 and 6.6 at different stages of cell density within 48 hours.
Material and methods Seconda.ry cultures of embryonic rat fibrobla,sts were prepared according to earlier data (NEUPERT et aI. 1972). In the 1st subculture the monolayers were cultured in Demeter flasks (50 cm 2 bottom area) using Eagle basal medium at pH G.6 and 7.4 (0.03 m HEPES buffer; Serva, Heidelberg). Accord-
159
ing to the cell density desired the cultures were ma,intained for 2 to 6 days, change of the medium at 2 days intervals. If necessary the media were supplemented with 100 mg% of L-Iactate or 200 mg% of D,L-Iactate. For the next change 50,uCi of 35S-sulfate (Isocommerz, Dresden) were added to each 10 ml of the fresh media. Incubation was stopped after 12 to 48 hours. MPS-precipitation by means of CPC was done after a.lcohol precipitation (medium plus trypsinized cells) and papain digestion as earlier described (KITTLICK et 301.1972, I, III). The MPS were eluted in fractions at cellulose columns according to SVEJCAR and ROBERTSON (1967). MPS-concentration was ascertained by hexuronic acid determina,tion (BITTER and MUIR 1962). Equivalents of the eluted fractions were treated with 0.5 ml of methanol and 0.5 ml of soluene (Packard, Wien) at 60°C and supplemented with a scintillatio,n cocktail (7.0 g PPO, 0.3 g dimethyl-POPOP, 100 g naphthalene in 11 of dioxan). Radiosulfate incorporation was measured by use of Tricarb 2211 (Packard) at quench correction with automatic externa.l standard. Besides chondroitin the fractions 1 and 2 contained preponderately HA; 3 = HS, 4 = Ch-4-S, 5 = Ch-6-S and higher molecular HS, 6 = DS (KITTLICK 1975). In fraction 2 sulfate was indiscernible. Fraction 4 contained low amounts of MPS va,rying in quantity thus it rendered impossible to obtain exact values.
Results and discussion 35S-sulfate incorporation into the fractions 3, 5 and 6 (HS, Ch-6-S, DS) was checked at pH 6.6 and 7.4 for a period of 48 hours. The results were dependent on cell density. In proliferating cultures (non-confluent cultures) the rise in specific activity was linear (cpm/,ug MPS; fig. la-b). In the stationary phase (confluent cultures) the specific activity demonstrated a balance of synthesis and breakdown after 24 hours (fig. 1c-d; cpo FRATANTONI et aI. 1968). Similar behaviour was observed in our experiments with an additive of 100 mg% L-Iactate in the culture medium at pH 7.4 and 6.6. ~
~
;tigm'S
1500
1000
a
~g MPS
6 5
1500 ..1.J1 5
~
6
500 3
3 24
7500
c
48 h
24
48 h
Q
7500 6 ~
...!.dQ 6
5
2500
5
3
3 48 h
48
h
Fig. 1. Rise of MPS specific activity dependent on the incubation period. Underlined numbers: cell count per flask at the beginning and at tbe end of incubation ( x 1(8); a, c = pH 6.6; b, d = pH 7.4. 3 = heparan sulfate, 5 = chondroitin-6-sulfate, 6 = dermatan sulfate. Example 1: a-b; example 2: c-d.
160
Specific activity and thus also the turnover rate are relatively low in HS (3). The specific activities observed in Ch-6-S (5) and DS (6) are markedly higher and similar to each other. The degree of hybridization of these 2 substances was not studied in our experiments. By fig. 1 it becomes evident that the specific activity of DS is higher than that of Ch-6-S at pH 7.4. In very dense cultures this difference was very expressive at pH 6.6 in a series of experiments (see KITTLICK et al. 1972, III). Occasionally it also occurred at cell densities of 1.5 x 106 cells per flask. This enhanced specific activity of DS in comparison with Ch-6-S is represented by regression lines for experiments with lactate additive (fig. 2 b) and without lactate (fig. 2a). The values were obtained after 48 hours of incubation. The course of the curves remained qualitatively unchanged following addition of 100 mg% of L-lactate, the labelling index DS/Ch-6-S in both pH ranges, however, was enhanced in favour of accelerated DS-turnover. Summarizing the results of this study it can be said that linear rise of 35S_ -sulfate incorporation is to be expected up to cell densities of about 2 x 106 cells per flask for 48 hours. SAl61
s:Al5f
2 pH 66 r = 0,23 (13) pH 14 r
2 3
2
5 .10 6 cells/ flask
3
-----
pH 6.6
r = 0,10 181
- - --.
2
i
3
= - 0,07 117)
pH 74 r=-O,32 (7)
, 4
Fig. 2. Quotient of the specific activities of fraction 6 (dermatan sulfate) and 5 (chondroitin-6 sulfate) dependent on cell density. a = without lactata: b = 100 mg% L-lachte. --------- pH 7.4, - - pH 6.6. In brackets: number of experiments.
Literature BITTER, T., and H. MUIR, A modified uronic acid carbazol reaction. Analyt. Biochem. 4, 330-334 (1962). FRATANTONI, J. C., C. W. HALL and E. F. NEUFELD, The defect in Hurler's and Hunter's syndromes: faulty degradation of mucopolysaccharide. Proc. atl. Acad. Sci. (U.S.) 60, 699-706 (1968). KITTLICK, P. D., G. NEUPERT and K. J. STILLER, Modification of the synthesis of acid mucopolysaccharides in fibroblast cultures. I. Chemical methods and changes in the pattern of distribution of mucopolysaccharides in the 1 st subculture. Exp. Path. 7, 7-18 (1972). III. Meta,bolic heterogeneity of acid mucopolysaccharides in fibrobla,st cultures of different pH values. Exp. Path. 7, 109-116 (1972). - - and F. BOLCK, MPS synthesis in fibroblast cultures. 1. Influence of cell density, pH-value and lactate concentration. Exp. Path, 12, 149-158 (1976). 3. in press, Exp. Path. (1976), 11
Exp. Path. Bd. 12
161
KITTLICK, P. D., The acid mucopolysaccharides in seconda,ry cultures of embryonic rat fibroblasts (Short communication). Exp. Path., 10, 359-363 (1975). NEUPERT, G., P. D. KITTLICK and K. J. STILLER, Modification of the synthesis of acid mucopolysaccharides in fibroblast cultures. II. Biological characteristics of a defined secondary fibroblast monolayer culture. Exp. Path. 7, 19-28 (1972). SVEJCAR, J., and W. VAN B. ROBERTSON, Micro separation and determination of mammalian acidic glycosa.minoglycans. Analyt. Biochem. 18, 333-350 (1967). Author's address: Dr. P. D. KITTLICK, Pathologisches Institut der Friedrich-Schiller-Universitat, DDR - 69 Jena, Ziegelmiihlenweg 1.
162
Acid mucopolysaccharides in fibroblast cultures 2. 3S S-sulfate incorporation kinetics in dependence on pH-value and cell density By P. D. KITTLICK, G. NEUPERT, K. J. STILLER and F. BOLCK With 2 figures (Received July 15, 1975) Key-words: acid mucopolysaccha,rides (MPS); 3sS-su lfate incorpora,tion; cell culture; cell density; pH-value; chondroitin sulfate; heparan sulfate; dermatan sulfate; lactate concentration; liquid scintillation; fibroblasts
Summary Cultures of embryonic rat fibroblasts were incubated with 3sS-sulfate at pH 6.6 and 7.4 (Eagle ba,saJ medium plus HEPES buffer) for 12 to 48 hours. The acid mucopolysaccharides wereisola,ted and fra,ctionated after the method of SVEJCAR and ROBERTSON. Sulfate incorporation was determined by liquid scintillation counting. Results: 1. In proliferating cultures linear rise of the specific activity of MPS (heparan sulfa,te, chondroitin-G-sulfate and dermatan sulfate) was observed for 48 hours. In stationary cultures balance of synthesis and breakdown occurs beginning with the 24th hour. The results apply to pH 6.G and 7.4. 2. The specific activity of dermatan sulfate is preponderating over that of chondroitin-G-sulfate. It is enha,nced by growing cell density. 3. Lactate addition to the culture medium at pH G.G a,nd 7.4 results in qualitatively similar effects. Abbreviations: Ch-6-S: chondroitin-G-slllfate CPC: cetylpyridinium chloride DS: dermata,n sulfate HA: hyaluronic a,cid HS: hepa.ran sulfa.te MPS: a,cid mucopolysaccharides, glycosaminoglycans S.A.: specific activity (cpm/fig MPS) The actual content of MPS in tissues and cell cultures represents a balance of de novo-synthesis and breakdown. In our previous paper we reported on the influence of pH value, lactate concentration and cell density on the content and distribution pattern of MPS in secondary cultures of embryonic rat fibroblasts (KITTLICK et a!. 1976, 1). In this as well as in two future papers we will describe the results of our simultaneous studies on 3sS-sulfate incorporation into HS, Ch-6-S and DS. Our findings give hints to what an extent de novo-synthesis and breakdown processes take part in the modification of the MPS-distribution pattern. The present study deals with the incorporation kinetics of 3sS-sulfate into the MPS-fractions HS, Ch-6-S and DS at pH 7.4 and 6.6 at different stages of cell density within 48 hours.
Material and methods Seconda.ry cultures of embryonic rat fibrobla,sts were prepared according to earlier data (NEUPERT et aI. 1972). In the 1st subculture the monolayers were cultured in Demeter flasks (50 cm 2 bottom area) using Eagle basal medium at pH G.6 and 7.4 (0.03 m HEPES buffer; Serva, Heidelberg). Accord-
159
ing to the cell density desired the cultures were ma,intained for 2 to 6 days, change of the medium at 2 days intervals. If necessary the media were supplemented with 100 mg% of L-Iactate or 200 mg% of D,L-Iactate. For the next change 50,uCi of 35S-sulfate (Isocommerz, Dresden) were added to each 10 ml of the fresh media. Incubation was stopped after 12 to 48 hours. MPS-precipitation by means of CPC was done after a.lcohol precipitation (medium plus trypsinized cells) and papain digestion as earlier described (KITTLICK et 301.1972, I, III). The MPS were eluted in fractions at cellulose columns according to SVEJCAR and ROBERTSON (1967). MPS-concentration was ascertained by hexuronic acid determina,tion (BITTER and MUIR 1962). Equivalents of the eluted fractions were treated with 0.5 ml of methanol and 0.5 ml of soluene (Packard, Wien) at 60°C and supplemented with a scintillatio,n cocktail (7.0 g PPO, 0.3 g dimethyl-POPOP, 100 g naphthalene in 11 of dioxan). Radiosulfate incorporation was measured by use of Tricarb 2211 (Packard) at quench correction with automatic externa.l standard. Besides chondroitin the fractions 1 and 2 contained preponderately HA; 3 = HS, 4 = Ch-4-S, 5 = Ch-6-S and higher molecular HS, 6 = DS (KITTLICK 1975). In fraction 2 sulfate was indiscernible. Fraction 4 contained low amounts of MPS va,rying in quantity thus it rendered impossible to obtain exact values.
Results and discussion 35S-sulfate incorporation into the fractions 3, 5 and 6 (HS, Ch-6-S, DS) was checked at pH 6.6 and 7.4 for a period of 48 hours. The results were dependent on cell density. In proliferating cultures (non-confluent cultures) the rise in specific activity was linear (cpm/,ug MPS; fig. la-b). In the stationary phase (confluent cultures) the specific activity demonstrated a balance of synthesis and breakdown after 24 hours (fig. 1c-d; cpo FRATANTONI et aI. 1968). Similar behaviour was observed in our experiments with an additive of 100 mg% L-Iactate in the culture medium at pH 7.4 and 6.6. ~
~
;tigm'S
1500
1000
a
~g MPS
6 5
1500 ..1.J1 5
~
6
500 3
3 24
7500
c
48 h
24
48 h
Q
7500 6 ~
...!.dQ 6
5
2500
5
3
3 48 h
48
h
Fig. 1. Rise of MPS specific activity dependent on the incubation period. Underlined numbers: cell count per flask at the beginning and at tbe end of incubation ( x 1(8); a, c = pH 6.6; b, d = pH 7.4. 3 = heparan sulfate, 5 = chondroitin-6-sulfate, 6 = dermatan sulfate. Example 1: a-b; example 2: c-d.
160
Specific activity and thus also the turnover rate are relatively low in HS (3). The specific activities observed in Ch-6-S (5) and DS (6) are markedly higher and similar to each other. The degree of hybridization of these 2 substances was not studied in our experiments. By fig. 1 it becomes evident that the specific activity of DS is higher than that of Ch-6-S at pH 7.4. In very dense cultures this difference was very expressive at pH 6.6 in a series of experiments (see KITTLICK et al. 1972, III). Occasionally it also occurred at cell densities of 1.5 x 106 cells per flask. This enhanced specific activity of DS in comparison with Ch-6-S is represented by regression lines for experiments with lactate additive (fig. 2 b) and without lactate (fig. 2a). The values were obtained after 48 hours of incubation. The course of the curves remained qualitatively unchanged following addition of 100 mg% of L-lactate, the labelling index DS/Ch-6-S in both pH ranges, however, was enhanced in favour of accelerated DS-turnover. Summarizing the results of this study it can be said that linear rise of 35S_ -sulfate incorporation is to be expected up to cell densities of about 2 x 106 cells per flask for 48 hours. SAl61
s:Al5f
2 pH 66 r = 0,23 (13) pH 14 r
2 3
2
5 .10 6 cells/ flask
3
-----
pH 6.6
r = 0,10 181
- - --.
2
i
3
= - 0,07 117)
pH 74 r=-O,32 (7)
, 4
Fig. 2. Quotient of the specific activities of fraction 6 (dermatan sulfate) and 5 (chondroitin-6 sulfate) dependent on cell density. a = without lactata: b = 100 mg% L-lachte. --------- pH 7.4, - - pH 6.6. In brackets: number of experiments.
Literature BITTER, T., and H. MUIR, A modified uronic acid carbazol reaction. Analyt. Biochem. 4, 330-334 (1962). FRATANTONI, J. C., C. W. HALL and E. F. NEUFELD, The defect in Hurler's and Hunter's syndromes: faulty degradation of mucopolysaccharide. Proc. atl. Acad. Sci. (U.S.) 60, 699-706 (1968). KITTLICK, P. D., G. NEUPERT and K. J. STILLER, Modification of the synthesis of acid mucopolysaccharides in fibroblast cultures. I. Chemical methods and changes in the pattern of distribution of mucopolysaccharides in the 1 st subculture. Exp. Path. 7, 7-18 (1972). III. Meta,bolic heterogeneity of acid mucopolysaccharides in fibrobla,st cultures of different pH values. Exp. Path. 7, 109-116 (1972). - - and F. BOLCK, MPS synthesis in fibroblast cultures. 1. Influence of cell density, pH-value and lactate concentration. Exp. Path, 12, 149-158 (1976). 3. in press, Exp. Path. (1976), 11
Exp. Path. Bd. 12
161
KITTLICK, P. D., The acid mucopolysaccharides in seconda,ry cultures of embryonic rat fibroblasts (Short communication). Exp. Path., 10, 359-363 (1975). NEUPERT, G., P. D. KITTLICK and K. J. STILLER, Modification of the synthesis of acid mucopolysaccharides in fibroblast cultures. II. Biological characteristics of a defined secondary fibroblast monolayer culture. Exp. Path. 7, 19-28 (1972). SVEJCAR, J., and W. VAN B. ROBERTSON, Micro separation and determination of mammalian acidic glycosa.minoglycans. Analyt. Biochem. 18, 333-350 (1967). Author's address: Dr. P. D. KITTLICK, Pathologisches Institut der Friedrich-Schiller-Universitat, DDR - 69 Jena, Ziegelmiihlenweg 1.
162