J. Biol. Chem., Vol. 255, Issue 21, 10091-10099, 11, 1980

The effect of cell density on net rates of glycosaminoglycan synthesis and secretion by cultured rat fibroblasts

L Hronowski and TP Anastassiades

Net rates of synthesis and secretion into the medium for hyaluronic acid and chondroitin sulfate have been calculated at varying cell densities of fibroblastic cell cultures from several tissues of the neonatal rat, utilizing an improved technique for glycosaminoglycan microquantitation. The incorporation of radioactivity from [3H]glucosamine and 35SO4 into the separated glycosaminoglycans from the cell surface and the cell pellet have also been estimated at varying cell densities. For rat muscle, skin, and heart fibroblasts, the net rate of hyaluronic acid synthesis per cell per day decreased rapidly with increasing cell densities, and the data best fit a linear relationship if plotted on semilog coordinates. The relationship of cell density to net chondroitin sulfate synthesis is more complex, but in the medium fraction a linear relationship is most closely approximated by plotting the data on linear coordinates. Glucose supplementation experiments and comparative rates of uptake of glucose from the medium by the various lines indicate that glucose depletion had only minor effects on the net synthesis and secretion of medium glycosaminoglycans. The y intercepts of linear plots of net glycosaminoglycan synthesis with varying cell density can be considered to reflect rates of synthesis and secretion at infinitely low cell densities. Under defined culture conditions, this value may be useful in characterizing the intrinsic synthetic capability of fibroblastic lines independent of the modifying influence of cell density.
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