Carmen Rossoni

Separation of N1- and N8-acetylspermidine isomers by reversed-phase column liquid chromatography after derivatization with dansyl chloride

The separation of dansyl derivatives of N1- and N8-acetylspermidine by reversed-phase column liquid chromatography is reported. The influence of organic solvents on the retention of acetylspermidines was studied. Best resolutions were achieved using a C18 column and a ternary mobile phase composed of water, methanol and acetonitrile. The precolumn derivatization method permitted the detection of picomole quantities. A method for the determination of acetylspermidines in rat tissues is described.

Effects of dexamethasone on spermidine N1-acetyltransferase and ornithine decarboxylase activities in rat spleen

Treatment of rats with the glucocorticoid dexamethasone causes an increase in the activity of cytosolic spermidine N1-acetyltransferase both in the spleen and thymus, but not, however, in liver, kidney or lung. The induced spermidine N1-acetyltransferase activity in the spleen catalyses acetylation of spermidine as well as spermine and sym-norspermidine, but not of diamines and histones. The enzyme induction depends on the dose of dexamethasone, and is suppressed by cycloheximide, which suggests that de novo protein synthesis is required for the action of this glucocorticoid. N1-acetylspermidine accumulates in the spleen after dexamethasone treatment, while spermidine progressively decreases and is partly converted into putrescine, the content of which transiently increases. In accordance with previous reports, dexamethasone was found to cause a rapid and large fall in the activity of spleen ornithine decarboxylase which was effected via the appearance of an inhibitor of the enzyme. Glucocorticoids exert large catabolic effects on lymphoid tissues, and further selectively affect the activities of spermidine N1-acetyltransferase and ornithine decarboxylase in the thymus and spleen. These latter selective responses may represent an important early event in lymphoid tissue response to glucocorticoid hormones.

Modulation of the induction of ornithine decarboxylase by some opioid receptor agonists in immune cells and cardiomyocytes.

The ability of natural and synthetic opioids to modulate the induction of ornithine decarboxylase (ODC) was investigated in immune cells and cardiomyocytes in culture. In particular, Leu-enkephalin, which shows preference for δ-receptors, enhanced ODC activity in both thymocytes and cardiomyocytes, whereas the effect of U-50488H, a synthetic κ-selective agonist, was cell-specific. In thymocytes, U-50488H markedly inhibited the induction of the enzyme elicited by the mitogen concanavalin A (Con A) or by a combined treatment with PMA and A23187, and also reduced basal ODC activity. However the drug did not affect ODC induced by other stimuli. The inhibition of the induction of ODC activity was accompanied by a reduction of ODC mRNA level and an acceleration of ODC turnover. The action of U-50488H in thymocytes does not appear to be mediated by κ or other classical opioid receptors lacking both stereospecificity and antagonist sensitivity, but may involve a pertussis toxin-sensitive G protein. Splenocytes also showed the ODC inhibiting effect of U-50488H, although they were less sensitive compared to thymocytes. In contrast, U-50488H enhanced ODC activity in cardiomyocytes and this effect was blocked by a specific κ-antagonist. In conclusion, these results indicate that some opioid agonists can modulate ODC expression in non neural cells. In particular, κ-opioid receptors may be involved in the U-50488H action in cardiomyocytes, and a distinct site, linked to inhibition of cell proliferation, may operate in immune cells.

Effect of adrenergic stimulation on ornithine decarboxylase activity in the rat spleen

The effect of a single administration of catecholamines on ornithine decarboxylase activity and polyamine biosynthesis in the rat spleen was investigated. Isoproterenol elicited a dose-dependent increase in spleen ODC activity which reached a maximum 4 hr after the administration of the drug. Putrescine content was also found to increase within a few hours, whereas S-adenosylmethionine decarboxylase activity and spermidine and spermine levels did not change significantly. Adrenaline and noradrenaline proved to be even more effective in increasing splenic ODC activity than isoproterenol. alpha- and beta-adrenergic antagonists prevented the ODC increase by catecholamines to a different extent.

Post-transcriptional inhibition of ornithine decarboxylase induction by zinc in a difluoromethylornithine resistant cell line

Addition of Zn2+ to cell medium inhibited the induction of ornithine decarboxylase (ODC) activity in ODC overproducing L1210-DFMOr cells. A significant effect was observed at a concentration as low as 0.01 mM, however, a more marked inhibition was caused by the addition of 0.1 mM Zn2+. The inhibition of the induction of ODC activity was accompanied by a proportional decrease in the content of immunoreactive ODC protein, whereas the level of ODC mRNA, determined by a solution hybridization RNase protection assay, was not affected significantly. Instead, some acceleration of ODC turnover was observed. The addition of 0.1 mM Co2+ or Mn2+, but not of other divalent metal ions, also inhibited ODC induction; differently from Zn2+ however, these metals affected cell viability and/or cell growth. Removal of endogenous Zn2+ by a chelator also provoked a strong decrease of ODC induction, which was reversed by Zn2+. However, addition of Zn2+ in excess of the chelator proved to be markedly inhibitory. These results indicate that both a restricted Zn2+ availability and an enhanced presence of the metal can inhibit the induction of ODC in L1210-DFMOr cells.

Inhibition of the expression of ornithine decarboxylase by some Κ-opioidergic receptor ligands in difluoromethylornithine-resistant L1210 cells

In difluoromethylornithine resistant L1210 cells stimulated to growth from quiescence, the selective kappa-opioidergic agonist trans-(+/-)-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneaceta mid e (U-50488H) caused a dose dependent inhibition of the induction of ODC activity, with a half-maximal effect at about 1 microM. U-50488H also provoked reduction of ODC mRNA level and increase of ODC turnover, as well as inhibition of cell growth. U-69593, another kappa-selective agonist, was only slightly effective. The action of U-50488H on ODC induction was not blocked by naloxone, beta-chlornaltrexamine or by the kappa-selective opioid antagonists Mr1452 and nor-binaltorphimine (nBNI). Actually Mr1452 and nBNI exerted some inhibitory effect. Furthermore, the separated enantiomers (+) and (-) of U-50488H were similarly effective. The (-)cis-(1S,2R)-U50488 stereoisomer, exhibiting low affinity for kappa and high affinity for sigma receptors and carbetapentane, another sigma ligand, also inhibited ODC induction, although less effectively than U-50488H. None of several other opioid ligands tested had significant effects on ODC induction. In conclusion, the inhibition of ODC expression by U-50488H does not involve classical, enantiospecific opioid receptors; rather, these results suggest the involvement of a distinct site of action linked to inhibition of lymphoid cell proliferation.

Inhibition of the expression of ornithine decarboxylase by haloperidol in difluoromethylornithine-resistant leukemia cells☆

In difluoromethylornithine-resistant L1210 cells stimulated to grow from quiescence, haloperidol caused an early and dose-dependent inhibition of the induction of ornithine decarboxylase (ODC) activity, with an IC50 of 3.5 microM. This effect was accompanied by a reduction in the ODC mRNA level and inhibition of cell growth. Other sigma ligands of different chemical classes inhibited the induction of ODC activity, whereas sulpiride, a dopamine antagonist devoid of sigma-binding affinity, was ineffective. These results indicate that the inhibition of ODC expression may be an early event involved in the antiproliferative response of leukemia cells to haloperidol.

Zinc can influence ornithine decarboxylase activity in rat thymus cells

SummaryThe thymus of young rats contained a high basal activity of ornithine decarboxylase (ODC). Treatment with zinc sulphate caused a slight increase of thymic ODC activity within 6 hours and a more marked enhancement (three-fold) in the spleen 24 h after treatment. In spite of the high activity of thymic ODCin vivo, ODC was not detectable in primary cultures of rat thymocytes, but was early and largely induced after treatment with Concanavalin A (Con A). The presence of 0.1 mM zinc in the medium increased the response of ODC to Con A. This effect of zinc in mitogen activated thymocytes may be due to the stabilization of ODC, which was found to decay with a half life of 65 min after the block of protein synthesis with cycloheximide. On the contrary in absence of zinc the half life of the enzyme was 40 min, as in the rat thymus in vivo.Zinc alone, at 0.1 mM concentration, did not affect ODC activity in resting thymocytes during the early times, but the metal was able to cause an increase of the enzyme activity after 4–6 days of culture. Other heavy metals such as mercury, cadmium and copper provoked a late increase of ODC activity, but their action was evident only at dosages which were toxic for the cells.