A comparison of the uptake, metabolism, and action ot cyclic adenine nucleotides in cultured hepatoma cells

Abstract

Intracellular radioactivity following incubation of HTC or RLC cells in [ 3H]cAMP exceeds that following incubation in either [ 3H]mono- or dibutyryl cAMP by 30-fold, yet little [ 3H]cAMP is found within the cells. Even at early times (30 min) the label derived from [ 3H]cAMP is predominantly found in ADP or ATP, suggesting it mostly enters the cell as the nucleoside. Significant intracellular concentrations of monobutyryl cAMP (2–10 μ m) result from incubation of both cell lines in either N 6 mono- or dibutyryl cAMP. A very small percentage of this label is in cAMP, and within 2 h of incubation > 65% of the label is again found in ADP or ATP. Liver cytosol contains three major cAMP-dependent protein kinases, designated A, B, and C, as resolved by DEAE-Sephadex chromatography. cAMP is the most effective in vitro activator (10- to 16-fold stimulation) of kinases A and B, the preponderant forms, in the order cAMP > N 6 monobutyryl cAMP ⋙ dibutyryl cAMP. Kinase C, a minor fraction, was stimulated two to threefold with the order cAMP ≥ N 6 monobutyryl cAMP > dibutyryl cAMP. HTC and RLC cell cytosol protein kinase has Chromatographic and cyclic nucleotide activation properties similar to those of liver fraction C. The activation state of the protein kinases of HTC and RLC cells incubated in the various cyclic nucleotides was also studied. The ability of such nucleotides to occupy regulatory protein binding sites in intact cells (as determined by the inhibition of subsequent in vitro binding of [ 3H]cAMP) was of the order N 6 monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. Correspondingly, the ratio of basal protein kinase activity in cyclic nucleotide treated:control cells was higher in cells incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP. This in vivo activation suggests that little additional stimulation would be obtained by adding cAMP to extracts prepared from such cells. This activation can be expressed as the ratio − cAMP: + cAMP (a ratio of 1 being maximal activation). The highest such ratio was seen in cells which had been incubated in monobutyryl cAMP > dibutyryl cAMP > cAMP > untreated cells. The studies indicate that all three cyclic nucleotides are capable of activating protein kinase in intact RLC and HTC cells; however the monobutyryl derivative is the most effective, and the degree of stimulation is greater in RLC than in HTC cells. RLC cell tyrosine aminotransferase activity is increased two to threefold by butyrylated cAMP derivatives (but not by cAMP) whereas the HTC cell enzyme is not induced. The rate of replication of both lines is unaltered by the butyrylated compounds. Since HTC and RLC cells accumulate and metabolize cAMP and its derivatives equally, and since they both contain a protein kinase with similar in vivo and in vitro activation properties, it is suggested that the effects of butyrylated cAMP derivatives on cell replication and tyrosine aminotransferase induction are mediated separately, either by distinct protein kinases, or at a point distal to protein kinase, or by a mechanism independent of protein kinase.