Increase In Polyamine Levels Research Articles

Polyamine levels as related to growth, differentiation and senescence in protoplast-derived cultures of Vigna aconitifolia and Avena sativa.

We have previously reported that aseptically cultured mesophyll protoplasts of Vigna divide rapidly and regenerate into complete plants, while mesophyll protoplasts of Avena divide only sporadically and senesce rapidly after isolation. We measured polyamine titers in such cultures of Vigna and Avena, to study possible correlations between polyamines and cellular behavior. We also deliberately altered polyamine titer by the use of selective inhibitors of polyamine biosynthesis, noting the effects on internal polyamine titer, cell division activity and regenerative events. In Vigna cultures, levels of free and bound putrescine and spermidine increased dramatically as cell division and differentiation progressed. The increase in bound polyamines was largest in embryoid-forming callus tissue while free polyamine titer was highest in root-forming callus. In Avena cultures, the levels of total polyamines decreased as the protoplast senesced. The presence of the inhibitors alpha-difluoromethyl-arginine (specific inhibitor of arginine decarboxylase), alpha-difluoromethylornithine (specific inhibitor of ornithine decarboxylase) and dicyclohexylamine (inhibitor of spermidine synthase) reduced cell division and organogenesis in Vigna cultures. Addition of low concentration of polyamines to such cultures containing inhibitors or removal of inhibitors from the culture medium restored the progress of growth and differentiation with concomitant increase in polyamine levels.

Developmental regulation of polyamine metabolism in growth and differentiation of carrot culture

Polyamine levels and the activities of two polyamine biosynthetic enzymes, arginine decarboxylase (EC 4.1.1.19) and S-adenosylmethionine decarboxylase (EC 4.1.1.50), were determined during somatic embryogenesis of carrot (Daucus carota L.) cell cultures. Embryogenic cultures showed severalfold increases in polyamine levels over nondifferentiating controls. A mutant cell line that failed to form embryos but grew at the same rate as the wild-type line also failed to show increases in polyamine levels, thus providing evidence that this increased polyamine content was in fact associated with the development of embryos. Furthermore, inhibition of these increases in polyamines caused by drugs inhibited embryogenesis and the effect was reversible with spermidine. The activities of arginine decarboxylase and Sadenosylmethionine decarboxylase were found to be suppressed by auxin; however, the specific effects differed between exogenous 2,4-dichlorophenoxyacetic acid and endogenous indole-3-acetic acid. The results indicate that increased polyamine levels are required for cellular differentiation and development occurring during somatic embryogenesis in carrot cell cultures.

Progressive increase in polyamine levels in 9L cells in vitro during the cell cycle: comparison between cells isolated by centrifugal elutriation and cells grown in synchrony.

Centrifugal elutriation was used to separate 9L rat brain tumour cells into fractions enriched in the G1, S, or G2/M phases of the cell cycle. Cells enriched in early G1 phase were recultured, grown in synchrony, and harvested periodically for analysis of their DNA distribution and polyamine content. Mathematical analysis of the DNA distributions indicated that excellent synchrony was obtained with low dispersion throughout the cell cycle. Polyamine accumulation began at the time of seeding, and intracellular levels of putrescine, spermidine, and spermine increased continuously during the cell cycle. In cells in the G2/M phase of the cell cycle, putrescine and spermidine levels were twice as high as in cells in the G1 phase. DNA distribution and polyamine levels were also analysed in cells taken directly from the various elutriation fractions enriched in G1, S, or G2/M. Because we did not obtain pure S or G2/M populations by elutriation or by harvesting synchronized cells, a mathematical procedure--which assumed that the measured polyamine levels for any population were linearly related to the fraction of cells in the G1, S, and G2/M phases times the polyamine levels in these phases and that polyamine levels did not vary within these phases--was used to estimate 'true' phase-specific polyamine levels (levels to be expected if perfect synchrony were achieved). Estimated 'true' phase-specific polyamine levels calculated from the data obtained from cells either sorted by elutriation or obtained from synchronously growing cultures were very similar.

Beta-adrenergic stimulation of Ca2+ fluxes, endocytosis, hexose transport, and amino acid transport in mouse kidney cortex is mediated by polyamine synthesis.

We recently found that the beta-adrenergic agonist 1-isoproterenol evokes a rapid (less than 5 min) Ca2+- and receptor-dependent stimulation of endocytosis, hexose transport, and amino acid transport in mouse renal cortex involving proximal tubule cells. This response is associated with increased Ca2+ fluxes and a mobilization of mitochondrial calcium, suggesting that stimulus-response (stimulus-"transport") coupling is mediated by cytosolic Ca2+. We show here that 1 microM isoproterenol evokes a rapid (less than 60 sec) transient increase in the activity of ornithine decarboxylase followed by an early (less than 2 min) sustained increase in putrescine, spermidine, and spermine concentrations in mouse kidney cortex slices in vitro. Small doses of isoproterenol (down to 24 nmol/kg) elicited a rapid (less than 2 min) increase in polyamines in vivo. The ornithine decarboxylase inhibitor alpha-difluoromethylornithine (5 mM) suppressed the testosterone-induced increase in polyamine levels and rates of endocytosis, hexose transport, and amino acid transport, measured by horseradish peroxidase, [14C]aminoisobutyric acid, and deoxy[3H]glucose uptake. alpha-Difluoromethylornithine also blocked the isoproterenol-induced increase in 45Ca influx and efflux and 45Ca redistribution; 0.5 mM putrescine nullified alpha-difluoromethylornithine inhibition and restored the increment in polyamines, 45Ca fluxes, endocytosis, hexose transport, and amino acid transport. These data implicate polyamine synthesis in isoproterenol stimulation of Ca2+ fluxes and membrane transport processes and support a model for signal transduction and stimulus-response coupling in which ornithine decarboxylase activation and polyamine synthesis play a pivotal role in regulating Ca2+ fluxes. In this model the polyamines generate local Ca2+ signals by stimulating Ca2+ influx or mobilizing intracellular calcium (or both) through a cation exchange reaction.

Role of Polyamines in Expression of the Differentiated Phenotype of Chondrocytes: Effect of DL-α-Hydrazino-δ-Aminovaleric Acid (DL-HAVA), an Inhibitor of Ornithine Decarboxylase, on Chondrocytes Treated with Parathyroid Hormone1

Previously, it was demonstrated that parathyroid hormone (PTH) induces a series of events, successive increases of the level of cyclic AMP, the activity of ornithine decarboxylase [ODC; EC 4.1.1.17] and glycosaminoglycan synthesis, a characteristic chondrocyte phenotype, in rabbit costal chondrocytes in culture. Cyclic AMP analogues also increase ODC activity and glycosaminoglycan synthesis in these cells. Therefore, in the present work, the role of polyamines in expression of the differentiated phenotype of chondrocytes was investigated. DL-alpha-Hydrazino-delta-aminovaleric acid (DL-HAVA), a potent inhibitor of ODC, inhibited the increase in polyamine levels of quiescent cultures of chondrocytes treated with PTH. DL-HAVA also inhibited stimulation of expression of the differentiated phenotype of chondrocytes by PTH, as judged by decreases in glycosaminoglycan synthesis and metachromasia on toluidine blue staining. Similarly, DL-HAVA inhibited stimulation of expression of the differentiated phenotype of chondrocytes by dibutyryl cyclic AMP. The inhibitory effect on expression of the differentiated phenotype of chondrocytes by DL-HAVA was effectively diminished by addition of polyamines. Under the same conditions, PTH, DL-HAVA, or polyamines had little effect on DNA synthesis. These findings suggest that the rise in polyamine levels induced by PTH, which is mediated by an increase of cyclic AMP concentration, is essential for expression of the differentiated phenotype of chondrocytes.

Polyamines and Root Formation in Mung Bean Hypocotyl Cuttings

The effect of several polyamines (putrescine, spermidine, and spermine), their precursors (l-arginine and l-ornithine), and some analogs and metabolic inhibitors (l-canavanine, l-canaline, and methylglyoxal-bis [guanylhydrazone]) on root formation have been studied in mung bean (Vigna radiata [L.] Wilczek) hypocotyl cuttings.Exogenously applied polyamines did not promote adventitious root formation. Rooting was inhibited by l-canavanine and l-canaline, and this inhibition was reversed by the corresponding amino acids l-arginine and l-ornithine. Methylglyoxal-bis (guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase and polyamine biosynthesis, was also found to inhibit root formation. All compounds at concentrations of >10(-4) molarity completely inhibited natural root formation, whereas at <10(-5) molarity only the indole-butyric acid-induced root formation was inhibited.Indole-butyric acid-induced root formation was accompanied by a considerable increase in polyamine levels, more than 2-fold of the control. Whereas senescing (unrooted) cuttings evinced a rapid decline in polyamine content during 48 hours, indole-butyric acid treatment resulted in elevated levels of putrescine and increased putrescine to spermidine ratio. The changes in polyamines were dependent on indole-butyric acid concentration and were organ specific.

Inhibitors of polyamine biosynthesis. 9. Effects of S-adenosyl-L-methionine analogues on mammalian aminopropyltransferases in vitro and polyamine biosynthesis in transformed lymphocytes.

Seven analogues of S-adenosyl-L-methionine were studied as inhibitors or substrates for mammalian spermidine and spermine synthases. One of these, S-(5'-deoxy-5'-adenosyl)-(+/-)-1-methyl-3-(methylthio)propylamine (5), showed a unique spectrum of activities on the polyamine biosynthesis enzymes. It was an inhibitor of S-adenosyl-L-methionine decarboxylase from rat liver and spermine synthase from bovine brain and rat ventral prostate. This compound was a substrate for the spermidine synthases from bovine brain and rat ventral prostate but not a substrate for the spermine synthases from these same sources. At concentrations of 0.2 mM and higher, compound 5 blocked the increases in polyamine levels and in [3H]thymidine incorporation induced by concanavalin A in cultured mouse lymphocytes. At approximately a 0.5 mM concentration of 5, the cellular polyamine levels and the rate of thymidine incorporation were similar to those of the unstimulated lymphocytes. Lower concentrations of 5 (0.02-0.1 mM) produced a dose-dependent increase in thymidine incorporation. A dose-dependent decrease in the cellular polyamine levels was observed in the range of 0.05-0.5 mM of the inhibitor. These results suggest that the effects of 5 on transformed lymphocytes are complex and may not be solely due to the inhibition of polyamine biosynthesis by this compound.

Biphasic effect of polyamines on chromatin-bound histone deacetylase

Calf thymus chromatin preparations contain a bound histone deacetylase. The activity of the deacetylase is increased by addition to the reaction mixture of 0.1 to 1 m m polyamine. Further increase in polyamine levels cause a progressive inhibition of enzyme activity. This biphasic action was shown to result from two opposite activities. First, low levels of polyamines are able to release several-fold greater amounts of enzyme from the bound state to free solution. Second, the free enzyme activity is progressively inhibited by polyamines from 0.1 to 20 m m. The combination of the two activities accounts for a peak of activity at around 1 m m polyamine.

ChemInform Abstract: INHIBITORS OF POLYAMINE BIOSYNTHESIS. V‐ IN VIVO EFFECTS OF α‐METHYL‐(.+‐.)‐ORNITHINE AND α‐METHYL‐(.+‐.)‐ORNITHINE TERT‐BUTYL ESTER

AbstractDie Wirkung der Titelverbindungen auf die Lebensdauer und den Polyamidspiegel von Mäusen, die mit L‐1 210 lxmgh ‐ tischen Leukämie‐Zellen behandelt worden sind, wird untersucht.

Inhibitors of polyamine biosynthesis V: In vivo effects of alpha-methyl-(+/-)-ornithine and alpha-methyl-(+/-)-ornithine tert-butyl ester.

Abstract The effects of a-methyl-(±)-ornithine and its tert -butyl ester on the survival time and the levels of polyamines in spleen tissue of mice inoculated intraperitoneally with L-1210 lymphoid leukemic cells were studied in vivo . These compounds were administered at doses of 100–300 mg/kg from Day 1 of tumor inoculation until death. This treatment neither significantly increased survival time nor altered the increases in polyamine levels normally observed during tumor growth. A method was developed to determine polyamine levels in spleen tissue. The polyamines in the tissue extracts were converted to their 5-(dimethylamino)-1-naphthalenesulfonyl derivatives, and these derivatives were separated by TLC followed by high-pressure liquid chromatography on a bonded phase microparticle column. The in vivo metabolism of 1- 14 C- α -methyl-(±)-ornithim was studied and compared to that of 1- 14 C-(±)-ornithine. 1- 14 C- α -Methyl-(±)-ornithine was rapidly excreted after administration to mice inoculated with L-1210 leukemic cells. Within 24 hr after injection, over 70% of the radioactive dose was found in the urine, primarily as the unchanged compound. Less than 0.1% of the administered dose appeared as 14 C-labeled carbon dioxide during the 24 hr following injection. In contrast, 34% of the administered dose of 1- 14 C-(±)-ornithine was metabolized to labeled carbon dioxide and 28% was excreted in urine during the 24 hr following injection.

Enzymatic decarboxylation of α-methyl amino acids

The effects of a-methyl-(±)-ornithine and its tert-butyl ester on the survival time and the levels of polyamines in spleen tissue of mice inoculated intraperitoneally with L-1210 lymphoid leukemic cells were studied in vivo. These compounds were administered at doses of 100–300 mg/kg from Day 1 of tumor inoculation until death. This treatment neither significantly increased survival time nor altered the increases in polyamine levels normally observed during tumor growth. A method was developed to determine polyamine levels in spleen tissue. The polyamines in the tissue extracts were converted to their 5-(dimethylamino)-1-naphthalenesulfonyl derivatives, and these derivatives were separated by TLC followed by high-pressure liquid chromatography on a bonded phase microparticle column. The in vivo metabolism of 1-14C-α-methyl-(±)-ornithim was studied and compared to that of 1-14C-(±)-ornithine. 1-14C-α-Methyl-(±)-ornithine was rapidly excreted after administration to mice inoculated with L-1210 leukemic cells. Within 24 hr after injection, over 70% of the radioactive dose was found in the urine, primarily as the unchanged compound. Less than 0.1% of the administered dose appeared as 14C-labeled carbon dioxide during the 24 hr following injection. In contrast, 34% of the administered dose of 1-14C-(±)-ornithine was metabolized to labeled carbon dioxide and 28% was excreted in urine during the 24 hr following injection.