Cyclic nucleotide phosphodiesterase activity in normal and neoplastic rat mammary cells grown in monolayer culture.
The activity of cyclic 3':5'-nucleotide phosphodiesterase (PDE) (EC 3.1.4.17) was measured in cultured normal and neoplastic rat mammary epithelium. Total PDE activity in normal cells was 1.6 to 6 times higher than that in tumor cells over a concentration range of 0.01 to 1 mM cyclic adenosine 3':5'-monophosphate. PDE activity was distributed between the low-speed (4000 x g) particulate and supernatant fractions in both cell lines, with the particulate fraction possessing 60 to 70% of the total. Double reciprocal kinetic plots were nonlinear, suggesting the presence of high- and low-affinity PDE activities. Similar, but not identical biphasic curves obtained from both normal and neoplastic cells suggested that at least two different PDE activities were present in a membrane-bound as well as a soluble form. Apparent Michealis constants for the high-affinity enzyme ranged from 2 to 6 muM; the low-affinity enzyme was 1 mM. In the presence of 10 mM caffeine and at a substrate concentration of 1 muM, PDE activity was inhibited 40 and 80% of basal levels in normal and tumor cells, respectively. In general, the membrane-bound enzyme was inhibited to a greater extent than the soluble, regardless of the cell line examined. Although normal cells exhibited higher PDE activities in terms of total specific activity, when soluble activities were compared at low substrate concentrations, the opposite was the case. At a substrate concentration of 0.01 muM, normal cell, low-Km soluble specific activity was 40% less than comparable tumor cell activity. Our results support the contention that PDE is induced by its own substrate, cyclic adenosine 3':5'-monophosphate. In addition, they suggest that the low cyclic adenosine 3':5'-monophosphate steady-state levels characteristic of malignant cells are maintained by a soluble high-affinity isozyme of PDE.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000032:analysis / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology; D007700:Kinetics; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008325:Mammary Neoplasms, Experimental / Q000201:enzymology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*
D002110:Caffeine; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases