Abstract

A variety of cyclic nucleotide derivatives were tested for their ability to activate cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) and to induce tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) in intact Reuber H35 rat hepatoma cells. The ratio of protein kinase activity toward a mixed histone preparation ± cAMP in 10,000 x g supernatant fractions of H35 cells incubated with or without a number of different cyclic nucleotide derivatives was used as an index of the degree of protein kinase activation in the intact cells. Basal ratios of 0.3-0.4 were observed but the actual ratio of cAMP-dependent protein kinase in untreated cells was estimated to be 0.10-0.15, based on the use of the skeletal muscle inhibitor and the slight stimulatory effect of dilution. The increase in the protein kinase activity ratio generated by each cyclic nucleotide derivative preceded that in aminotransferase activity, a pattern to be expected if a cause-effect relationship exists. The dose-response relationships for a variety of these derivatives revealed a highly significant correlation between kinase activation and enzyme induction. Furthermore, the effects of cAMP itself and two derivatives on both processes were converted from weak to strong by addition of a phosphodiesterase inhibitor. One of the cyclic nucleotide derivatives tested, 8-parachlorophenylthio cAMP, exhibited dramatically higher potency (ED₅₀ for kinase activation and enzyme induction ≅ 2-3 µM) than N⁶,O²'-dibutyryl cAMP (ED₅₀ ≅ 100 µM). Sephadex G-100 chromatography of protein kinase activity was used to confirm the results of the ratio method. The results demonstrate that enzyme induction is tightly coupled to protein kinase activation and is consistent with the possibility that these two processes are causally related.