Characterization of serum-induced alterations in the cyclic AMP pathway in S49 lymphoma cells.

We have grown the 549 lymphoma cell line in a serum-free, chemically defined medium which contains casein, insulin, transferrin, testosterone, and linoleic acid.When grown in this medium, wild type 549 cells display a 2-3-fold increase in hormonal @-adrenergic, prostaglandin El) and nonhormonal (cholera toxin) stimulation of CAMP accumulation compared to cells grown in 10% horse serum.Wild type cells grown in serum-free medium contained 30% more 8-adrenergic receptors than did serum-grown cells, although the affinity of the receptors for [3H]dihydroalprenolo1 was unchanged.The 549 variants, UNC and kin-, failed to show the alterations in cholera toxin-or isoproterenolstimulated CAMP accumulation that were observed with wild type cells.lZ6I-labeled cholera toxin binding studies showed no change in the number or affinity of cholera toxin receptors in wild type or kin-cells grown under the two conditions.In cell homogenates, wild type cells grown in serum-containing medium had 28-45% higher activity of cyclic nucleotide phosphodiesterase than did cells grown in serum-free medium.Growth with or without serum produced no change in the K, values (14 and 0.25 p ~, respectively, for the two forms) of phosphodiesterase in these cells.Basal, NaF-, Gpp(NH)p-, and isoproterenol plus GTP-stimulated adenylate cyclase activities were similar in crude plasma membrane preparations from serum-free and serum-grown wild type cells.Horse serum contained a nondialyzable (greater than 8000 daltons), nonlipid factor which depressed the isoproterenol-stimulated CAMP accumulation of wild type 549 cells and which required 3-4 h to produce this effect.This factor appears to act via the nucleotide-coupling unit of the adenylate cyclase system and protein kinase, and ultimately results in an increase in cyclic nucleotide phosphodiesterase activity.We suggest that studies of the CAMP pathway in 549 cells should ideally be carried out under the less perturbing conditions offered by serum-free medium.