Modulation of insulin induced ornithine decarboxylase by putrescine and methylputrescines in H-35 hepatoma cells.
The effect of several methylputrescines on the activity of insulin-induced ornithine decarboxylase (ODC) was examined in H-35 hepatoma cells. The induction involved both protein and m-RNA synthesis. Actinomycin D inhibited ODC activity when given up to 1 h after insulin treatment. When added to the medium 2 h or 3 h after the insulin, the activity was increased 100% and 80% respectively. Insulin-induced ODC from H-35 cells had a biphasic half-life, a shorter one of 46 min and a longer one of 90 min. 1-Methylputrescine and 2-methylputrescine were found to be competitive inhibitors of the ODC from H-35 cells with Ki values of 2.8 and 0.1 mM respectively. Putrescine itself was found to have a Ki = 2.4 mM. N-Methylputrescine was a very poor inhibitor of the cell free ODC while 1,4-dimethylputrescine did not show any inhibitory effect. When cellular ODC activity was measured, the four methylputrescines assayed as well as putrescine entirely abolished its activity in the H-35 cells when given at a 1 mM concentration together with insulin. 1-Methylputrescine and 1,4-dimethylputrescine abolished 60% of the activity at a 0.1 microM concentration. All the methylputrescines given at 0.1 mM concentrations decreased the putrescine content of the stimulated cells to the levels found in quiescent cells, but only 1-methyl and 2-methylputrescines decreased spermidine and spermine content. 1,4-Dimethyl and 1-methylputrescines showed a strong inhibition of ODC synthesis, while the other diamines were less inhibitory. At concentrations that abolished ODC activity, 1,4-dimethylputrescine decreased 70% of the total immunoreactive ODC bands, while 1-methyl and 2-methylputrescine decreased them by 50%, and N-methylputrescine and putrescine decreased them by 20%. The lack of decrease in immuno-reactive ODC with the latter two compounds was mainly due to the appearance of immunoreactive degradation products of ODC of low molecular weight. Putrescine and N-methylputrescine affected protein synthesis to a small extent in stimulated cells, while 1-methylputrescine decreased it to the level of non-stimulated cells. Insulin (1 microM concentration) stimulated DNA synthesis in the cells, and this stimulation was doubled in the presence of 2-methylputrescine or putrescine. It can be concluded that, among the methylputrescines assayed, 2-methylputrescine was the best inhibitor of cell-free ODC activity, while 1,4-dimethylputrescine and 1-methylputrescine were the best inhibitors of cellular ODC activity.
Frydman
J
J
Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
Ruiz
O
O
Robetto
E
E
Dellacha
J M
JM
Frydman
R B
RB
D000818:Animals; D004247:DNA / Q000096:biosynthesis; D003609:Dactinomycin / Q000494:pharmacology; D004789:Enzyme Activation; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental; D009955:Ornithine Decarboxylase / Q000096:biosynthesis / Q000378:metabolism*; D014176:Protein Biosynthesis; D011700:Putrescine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D051381:Rats; D014407:Tumor Cells, Cultured
D007328:Insulin; D003609:Dactinomycin; D004247:DNA; D009955:Ornithine Decarboxylase; D011700:Putrescine