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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.

Hexamethylene bisacetamide (HMBA) stimulates Ca(2+) signals in murine erythroleukemia (MEL) cells serving as an important component of the HMBA-induced pathway that promotes differentiation to the erythroid phenotype. We observed that 1,6-diaminohexane (DAH) triggered a more rapid and robust increase in MEL cell Ca(2+) levels compared to HMBA and the monodeacetylated N-acetyl-1,6-diaminohexane (NADAH), and that polyamine deacetylase inhibition completely abolished the ability of HMBA and NADAH to induce Ca(2+) signals in MEL cells. Our work indicates that DAH mediates Ca(2+) signal propagation via its ability to activate inositol 1,4,5-trisphosphate (IP(3)) receptors, as we observed similar Ca(2+) release characteristics and heparin sensitivity of DAH and IP(3) in permeabilized MEL cells. Finally, we observed that the DAH-induced Ca(2+) release pathway robustly coupled to a Ca(2+) influx pathway that could be distinguished from thapsigargin-induced Ca(2+) influx by its unusual insensitivity to 2-aminoethoxydiphenyl borate.

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.

Metabolism of N1-acetylspermidine and N8-acetylspermidine in rat liver and kidney was studied in vivo. N1-Acetylspermidine was metabolized primarily to putrescine while N8-acetylspermidine underwent deacetylation to yield spermidine. The rate of metabolism of these two compounds was much greater than that of spermidine. The rate of metabolism of N8-acetylspermidine exceeded that of N1-acetylspermidine in both liver and kidney. These rapid rates of metabolism could at least in part account for the low levels of these two compounds found in tissues. The differences in routes of metabolism of N1-acetylspermidine and N8-acetylspermidine may indicate differences in cellular functions of the two compounds.

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.

The inhibitory effects of 7-[N-(3-aminopropyl)amino]heptan-2-one (APAH) on N8-acetylspermidine deacetylation were studied. In in vitro studies, APAH produced inhibition (apparent Ki of 0.18 microM) of N8-acetylspermidine deacetylation by the 100,000g supernatant fraction of rat liver. This apparent Ki was 60-fold less than the apparent Km (11 microM) for deacetylation of the substrate, N8-acetylspermidine, suggesting that APAH could be a potent, effective inhibitor in vivo. APAH was administered to mice by intraperitoneal injection at a dose of 200 mg/kg, and polyamine and acetylpolyamine levels in liver and spleen were measured. In tissues of control mice, N8-acetylspermidine was not detectable but increased to detectable levels 30-360 min after APAH treatment. These data are consistent with inhibition of the deacetylase by APAH. Increases in putrescine and N1-acetylspermidine levels occurred in liver after APAH treatment with increases in N1-acetylspermidine levels observed in spleen. In HeLa cells, a significant increase in N8-acetylspermidine was observed following 24 h exposure to 10 microM APAH while no change occurred in the acetylation level of HeLa cell histones. In contrast, 24 h exposure to 10 mM sodium butyrate produced no change in N8-acetylspermidine levels and an increase in the acetylation level of histones H4 and H2B. These results suggest that APAH has a relatively selective inhibitory effect on N8-acetylspermidine but not histone deacetylation. This is the first report of significant levels of N8-acetylspermidine in animal tissues and of the effects of in vivo inhibition of N8-acetylspermidine deacetylase.

The in vivo effects of phenobarbital on 32 P incorporation into rat liver acidic nuclear proteins were studied. Nuclei were isolated in citric acid and the acidic nuclear proteins were extracted with phenol. Electrophoretic fractionation of these proteins was achieved on 10% acrylamide gels containing 0.1% sodium dodecylsulfate at pH 7.2. A stimulation of 32 P incorporation into acidic nuclear proteins was observed within 2–6 h after administration of drug. A maximum, 48% greater than control level, was observed at 24 h after treatment. The electrophoretic profiles of the incorporated 32 P indicated that the stimulation may be selective for specific acidic nuclear proteins; however, the phosphorylation of all the acidic nuclear phosphoproteins seems to be stimulated to some degree. These results suggest that phosphorylation of acidic nuclear proteins may play a role in the effects of phenobarbital on liver growth or microsomal enzyme activity.

Reducing agents are commonly used to limit aggregation of acidic nuclear proteins in aqueous media. However, these agents interfere with protein determinations and thus make it difficult to continuously monitor protein concentration. In this study, acidic nuclear proteins were alkylated with iodoacetamide to eliminate the need for a reducing agent. The alkylated proteins can be easily analyzed; and furthermore, alkylation may enhance the resolution obtained by column chromatography or gel electrophoresis.