MECHANISM OF ENHANCED CYCLIC-AMP STIMULATION BY ISOPROTERENOL IN AGED HUMAN FIBROBLASTS

Human diploid lung fibroblasts (IMR-90) were used to investigate the reported increase in beta-adrenergic-stimulated cyclic adenosine 3', 5'-monophosphate (cAMP) levels in fibroblasts aged in culture. Under basal conditions cellular cAMP was 34.2 +/- 5.6 and 38.4 +/- 9.1 pmol/mg protein in early (PDL 22-24) and late (PDL 47-52) passage fibroblasts, respectively. Net release of cAMP from fibroblasts was 67.8 +/- 8.6 and 18.5 +/- 7.0 pmol/30 min/mg protein in early and late passage cultures, respectively. In confluent, early passage fibroblasts, cellular cAMP and net release of cAMP increased by 2.7-fold and 3.8-fold, respectively, after a 30 min incubation in 2-mu-M isoproterenol. In confluent late passage fibroblasts, isoproterenol incubation increased cellular cAMP and net release of cAMP by 7.8-fold and 26. 1 -fold, respectively. Adenosine failed to inhibit isoproterenol-induced stimulation of cAMP in early or late passage fibroblasts. There was no passage-related difference in basal, isoproterenol, or forskolin-stimulated adenylyl cyclase activity in crude fibroblast membrane preparations. The activity of cAMP-phosphodiesterase in sonicates of early and late passage IMR-90 was 9.61 +/- 1.15 and 5.81 +/- 1.11 pmol/min/mg protein respectively. Measurements of cAMP in subconfluent early passage fibroblasts indicated that mechanisms related to the reduced cell density in confluent late passage IMR-90 may, in part, account for the enhanced isoproterenol-induced cAMP levels observed in these cultures. The results suggest that the remainder of the enhanced cAMP response to isoproterenol of in vitro aged fibroblasts may be due to a lower cAMP phosphodiesterase activity in these cells.