Search

Spermidine is one of the simple polyamines found in cells of virtually all living organisms. It undergoes a metabolic conversion to N8-acetylspermidine catalyzed by an enzyme in cell nuclei and is converted back to spermidine by a deacetylase in the cytoplasm. In this study, two different mechanisms were used to produce an elevation in the level of N8-acetylspermidine in PC12 cells: inhibition of N8-acetylspermidine deacetylase and direct addition of N8-acetylspermidine to the cell culture. The increasing intracellular concentration of N8-acetylspermidine was accompanied by signs of PC12 cell differentiation including increased content of dopamine and morphological changes (neurite outgrowths), suggesting a strong and perhaps causal relationship among these effects. This effect on differentiation appears to be specific for N8-acetylspermidine as the addition of other polyamines including spermidine and N1-acetylspermidine did not elicit these changes. Nerve growth factor (NGF) and dexamethasone, commonly used inducers of differentiation in PC12 cells, produced differentiation without measurable changes in N8-acetylspermidine levels, suggesting that different (or multiple) mechanisms may be involved in these differentiation processes.

A selective inhibitor of N8-acetylspermidine deacetylase has been employed to study the role of N8-acetylspermidine deacetylation in the regulation of L1210 cell growth. This inhibitor, 7-[N-(3-aminopropyl) amino] heptan-2-one (APAH), was found to stimulate the growth of L1210 cells at concentrations between 10 microM and 0.5 mM. Maximum stimulation was seen at 100 microM, resulting in significantly increased rates of cell division and maximum cell density. N8-Acetylspermidine levels in L1210 cells were shown to increase significantly after the APAH treatment as would be expected for deacetylase inhibition. The effects of deacetylase inhibition were mimicked by addition of N8-acetylspermidine to the culture medium at concentrations greater than 1 mM as indicated by a subsequent increase in rate of cell growth and maximum cell density. The magnitudes of the increases in growth observed were not large, but this might be expected in cells that are already in a rapid growth phase. Other exogenously added polyamines including N1-acetylspermidine, spermidine, putrescine, and spermine did not stimulate cell growth. These data suggest that stimulation of cell growth occurs as a consequence of N8-acetylspermidine accumulation and N8-acetylspermidine deacetylase inhibition.

The in vitro enzymatic acetylation of the polyamines, spermidine and spermine, is described. The reaction is catalyzed by chromatin preparations from rat liver and kidney and is dependent on acetyl-CoA. Spermidine, spermine, and putrescine are each converted to the corresponding monoacetyl derivatives. s 0.5 values of 0.5 ± 0.1, 1.0 ± 0.1, and 2.6 ± 0.7 m m (mean ± standard deviation) were obtained for spermidine, spermine, and putrescine, respectively. These values for s 0.5 are similar to the concentrations of polyamines reported for tissues, and therefore, suggest the occurrence of polyamine acetylation in vivo . Evidence is also presented for the metabolism of acetylated polyamines by the 100,000 g supernatant fraction of rat liver. The physiological function of polyamine acetylation is unknown, but the possibility of an effect on the association of polyamines with nucleic acids is discussed.

The in vitro deacetylation of N8-acetylspermidine by an enzyme activity in rat tissues is described. This deacetylase activity occurs as a soluble, cytoplasmic enzyme in rat liver and was detected in the 100,000g supernatant fraction of all tissues examined. The highest specific activity was found in liver. Spleen, kidney, and lung were found to contain 20–50% of the activity in liver, while heart, brain, and skeletal muscle exhibited from 2 to 10% of the activity in liver. Serum contained only barely detectable levels of activity, much lower than any of the tissues studied. The in vitro metabolism of N1-acetylspermidine differed from that observed for N8-acetylspermidine and does not appear to involve a simple deacetylation reaction.

The specificity of N-acetylspermidine deacetylase from rat liver for the two naturally occurring forms of monoacetylated spermidine was studied. N8-Acetylspermidine is the preferred substrate in vitro for this enzyme, and, in fact, N1-acetylspermidine did not undergo deacetylation under the conditions used in this study. Thus N8-acetylspermidine is the more appropriate substrate for assaying N-acetylspermidine deacetylase activity.