P.D. Kittlick

The acid mucopolysaccharides (glycosaminoglycans) in secondary cultures of embryonic rat fibroblasts.

In secondary cultures of embryonic rat fibroblasts the protein-free acid mucopolysaccharides were separated by cetylpyridinium chloride fractionation and identified afterwards. The following acid mucopolysaccharides were found: hyaluronic acid, chondroitin, 2 heparan sulphate fractions, chondroitin-4-sulphate, chondroitin-6-sulphate, and dermatan sulphate.

Acid mucopolysaccharides in fibroblast cultures: 3.Influence of lactate on the MPS-distribution pattern and 35S-sulfate incorporation at different cell densities

Summary The influence of 100 mg % L-lactate in the culture media of monolayers of embryonic rat fibroblasts was studied at p H 7.4.Cell proliferation, amount of total MPS, MPS-distribution pattern, 35 S-sulfate incorporation (relative synthesis rate, specific activity, MPS-metabolism) were determined in their dependence on cell density.

Acid mucopolysaccharides in fibroblast cultures

From cells and culture media of embryonic rat fibroblasts (1st subculture) the acid mucopolysaccharides were isolated and fractionated. The per cent calculation of the 6 fractions was based on the content of glucuronic acid. The cultures were maintained as follows: 0.5 X 10(6) to 3 X 10(6) cells were examined in Demeter flasks at pH 7.4 or 6.6; lactate concentration was enhanced to 100 mg%. The amount of each fraction was correlated with the cell density (linear regression). The pH-value and lactate concentration in connection with cell density proved to be important factors in the modification of the MPS distribution pattern.

Hypoxia in fibroblast cultures: 2. Influence of pH on the distribution pattern of acid mucopolysaccharides

Secondary cultures of embryonic rat fibroblasts [NEUPERT et al., Exp. Path. 7, 19-28 (1972)] were cultured in 21% and 5% O2 concentration at pH 6.6 and 7.4 for 6 or 8 days. The acid mucopolysaccharides were isolated and fractionated by alcohol precipitation, papain digestion, CPC-precipitation and fractionation after the microtechniques of SVEJCAR and ROBERTSON [see KITTLICK and NEUPERT, Exp. Path. 7, 7-18 (1972)]. Cells and medium were investigated together. The results were related to cell density and compared with the glucose and lactate values [see KITTLICK and NEUPERT, Exp. Path. 10, 109-114 (1975)]. Concerning the question of interrelations of MPS-synthesis and glycolysis a survey on literature is given. Our own test results were as follows: 1. Hypoxia (5% O2) did not influence MPS-total synthesis. 2. Depending on cell density the individual MPS fractions were different in their reaction to hypoxia. 3. Hyaluronic acid (and heparan sulphate) in the MPS-pattern showed other behaviour than chondroitin sulphate and dermatan sulphate, respectively. 4. At low cell density hypoxia effectuated increase in hyaluronic acid and decrease in chondroitin sulphate and dermatan sulphate, respectively. 5. At high cell density hypoxia effectuated decrease in hyaluronic acid and increase in chondroitin sulphate and dermatan sulphate, respectively. Possible relationship to processes in the tissue is discussed.

Hypoxia in fibroblast cultures: 3. 35S-sulfate incorporation into acid mucopolysaccharides as influenced by 5% O2 hypoxia with simultaneous changes in the lactate- and H-ion concentrations

The influence of 5% O2 hypoxia on the 35S-sulfate incorporation into different mucopolysaccharide fractions was studied in monolayer cultures of embryonic rat fibroblasts before reaching the stationary phase. Besides the O2 concentration also the pH value and lactate concentration were varied in the experiments. The results are valid for proliferating cell cultures.

Acid mucopolysaccharides in fibroblast cultures: 2. 35S-sulfate incorporation kinetics in dependence on pH-value and cell density

Cultures of embryonic rat fibroblasts were incubated with 35S-sulfate at pH 6.6 and 7.4 (Eagle basal medium plus HEPES buffer) for 12 to 48 hours. The acid mucopolysaccharides were isolated and fractionated after the method of SVEJCAR and ROBERTSON. Sulfate incorporation was determined by liquid scintillation counting.

Clotting factors added to fibroblast cultures. Their action on glycosaminoglycans and other parameters.

To monolayer cultures of embryonic rat fibroblasts in the proliferative and stationary phase of growth there were given: thrombin, fibrinogen or fibrin supernatant, respectively. Their effects on cell proliferation, glucose consumption and glycosaminoglycans were recorded and observed to be more pronounced in serum-depleted and confluent cultures. Thrombin in serum-supplemented cultures was nearly ineffective. In serum-free stationary cultures glucose consumption, GAG concentration and, above all, hyaluronic acid were increased. Fibrinogen stimulated the metabolism of stationary fibroblasts (glucose, GAG, particularly hyaluronic acid) more strongly in serum-depleted medium. A number of protease inhibitors were ineffective in abolishing the fibrinogen action pointing to the efficacy of the intact fibrinogen molecule. The supernatant of the fibrinogen-thrombin-reaction, separated after 3 hours, likewise increased glucose consumption, GAG and hyaluronic acid concentration possibly due to effects of the fibrinopeptides A or B. However, contamination of fibrinogen with other active compounds cannot be excluded as yet. Surprisingly, fibrin generated on the fibroblast monolayer did not stimulate the cells. Therefore fixation of the active compounds of the fibrin supernatant (fibrinopeptides) during the process of fibrin polymerization has to be assumed. According to these observations thrombin, fibrinogen and components of the fibrin supernatant contribute to the increase of hyaluronic acid and cell activation in the oedematous phase of inflammation at sites free from fresh-formed fibrin.

Acid mucopolysaccharides in fibroblast cultures: 4. 35S-sulfate incorporation in dependence on pH-value cell density and lactate

UNLABELLED Cultures of embryonic rat fibroblasts were incubated with 35S-sulfate at pH 6.6 and 7.4 (Eagle medium, Hepes buffer) for 48 hours. The MPS were isolated and fractionated. Determination of hexuronic acid was done according to BITTER and MUIR, measuring of sulfate incorporation by liquid scintillation counting. RESULTS 1. HS, Ch-6-S and DS showed different synthesis rates. They rised at increasing cell density and were lower at pH 6.6 than at 7.4. 2. At pH 6.6 the sulfate incorporation rates (de novo-synthesis) of HS, Ch-6-S and DS are lowered. The percent increase of the MPS fractions at pH 6.6 and their specific activities are explained by individual differences in the unlabelled MPS pools as well as by differences in the rates of synthesis and catabolism. The cell density also has an influence on that. Increase of the MPS at pH 6.6 is by mainly due to an inhibition of catabolism. 3. Contrary to CH-6-S and DS heparan sulfate is catabolized more slowly at high cell densities. 4. The volume of the unlabelled MPS pool is modified by lactate at pH 6.6, e.g. it effectuates enhancement of the DS pool at low cell densities. De novo-synthesis and specific activity are scarcely influenced. CH-6-S and DS catabolism are considerably inhibited, their total amount is increased. The relative synthesis rate of DS is enhanced by lactate. 5. The possible importance of the results for inflammatory processes and wound healing is mentioned.

Hypoxia in fibroblast cultures. 4. Cell density, glucose utilization and acid mucopolysaccharides in 1% O2 concentration.

Secondary cultures of embryonic rat fibroblasts incubated in Eagle medium with Hepes buffer at pH 6.6 and 7.4 were maintained in hypoxia of 1% O2 for 48 hours. The acid mucopolysaccharides were isolated from medium plus cells and fractionated on cellulose microcolumns according to SVEJCAR and ROBERTSON (1967). Glucose utilization, lactate production, quantity of total MPS and changes in the MPS distribution pattern were correlated with cell density.

Hypoxia in fibroblast cultures. I. Changes in glucose consumption by 5 vol.-% oxygen concentration and reduced pH.

In secondary cultures of rat embryonic fibroblasts the glucose consumption and lactate concentration were comparatively investigated in 5 and 21 Vol.-% O2 environment at pH 6.6 and pH 7.4 (HEPES-buffer). The results were correlated with cell density. The following observations were made: 1. At pH 6.6 the rate of cell proliferation was reduced to 81%; by additional hypoxia it was reduced to 71%. 2. Increase in cell density effectuated decrease of glucose consumption and lactate production at pH 7.4 and pH 6.6 in 5% as well as in 21% O2 environment. 3. At pH 7.4 enhancement of glucose consumption and lactate production due to hypoxia can be observed only at low cell density. 4. At pH 6.6 respiration of fibroblasts was little influenced by 5% O2 environment. 5. Transition from pH 7.4/21% O2 to pH 6.6/5% O2 effectuated decrease in glucose consumption and lactate concentration in tissues in hypoxic environment. This suggests a pH-governed feedback mechanism. Abbreviations used in the text: c-AMP = cyclic adenosine monophosphate; MPS = acid mucopolysaccharides (glycosaminoglycans).