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DL-alpha-difluoromethyl[3,4-3H]arginine metabolism in tobacco and mammalian cells. Inhibition of ornithine decarboxylase activity after arginase-mediated hydrolysis of DL-alpha-difluoromethylarginine to DL-alpha-difluoromethylornithine.

ABSTRACT: DL-alpha-Difluoromethylarginine (DFMA) is an enzyme-activated irreversible inhibitor of arginine decarboxylase (ADC) in vitro. DFMA has also been shown to inhibit ADC activities in a variety of plants and bacteria in vivo. However, we questioned the specificity of this inhibitor for ADC in tobacco ovary tissues, since ornithine decarboxylase (ODC) activity was strongly inhibited as well. We now show that [3,4-3H]DFMA is metabolized to DL-alpha-difluoromethyl[3,4-3H]ornithine [( 3,4-3H]DFMO), the analogous mechanism-based inhibitor of ODC, by tobacco tissues in vivo. Both tobacco and mammalian (mouse, bovine) arginases (EC 3.5.3.1) hydrolyse DFMA to DFMO in vitro, suggesting a role for this enzyme in mediating the indirect inhibition of ODC by DFMA in tobacco. These results suggest that DFMA may have other effects, in addition to the inhibition of ADC, in tissues containing high arginase activities. The recent development of potent agmatine-based ADC inhibitors should permit selective inhibition of ADC, rather than ODC, in such tissues, since agmatine is not a substrate for arginase.

SUBMITTER: Slocum RD

PROVIDER: S-EPMC1135209 | biostudies-other | 1988 Oct

REPOSITORIES: biostudies-other

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Project description:Background:Polyamines stimulate DNA transcription and mRNA translation for protein synthesis in trophectoderm cells, as well as proliferation and migration of cells; therefore, they are essential for development and survival of conceptuses (embryo/fetus and placenta). The ovine conceptus produces polyamines via classical and non-classical pathways. In the classical pathway, arginine (Arg) is transformed into ornithine, which is then decarboxylated by ornithine decarboxylase (ODC1) to produce putrescine which is the substrate for the production of spermidine and spermine. In the non-classical pathway, Arg is converted to agmatine (Agm) by arginine decarboxylase (ADC), and Agm is converted to putrescine by agmatinase (AGMAT). Methods:Morpholino antisense oligonucleotides (MAOs) were designed and synthesized to inhibit translational initiation of the mRNAs for ODC1 and ADC, in ovine conceptuses. Results:The morphologies of MAO control, MAO-ODC1, and MAO-ADC conceptuses were normal. Double knockdown of ODC1 and ADC (MAO-ODC1:ADC) resulted in two phenotypes of conceptuses; 33% of conceptuses appeared to be morphologically and functionally normal (phenotype a) and 67% of the conceptuses presented an abnormal morphology and functionality (phenotype b). Furthermore, MAO-ODC1:ADC (a) conceptuses had greater tissue concentrations of Agm, putrescine, and spermidine than MAO control conceptuses, while MAO-ODC1:ADC (b) conceptuses only had greater tissue concentrations of Agm . Uterine flushes from ewes with MAO-ODC1:ADC (a) had greater amounts of arginine, aspartate, tyrosine, citrulline, lysine, phenylalanine, isoleucine, leucine, and glutamine, while uterine flushes of ewes with MAO-ODC1:ADC (b) conceptuses had lower amount of putrescine, spermidine, spermine, alanine, aspartate, glutamine, tyrosine, phenylalanine, isoleucine, leucine, and lysine. Conclusions:The double-knockdown of translation of ODC1 and ADC mRNAs was most detrimental to conceptus development and their production of interferon tau (IFNT). Agm, polyamines, amino acids, and adequate secretion of IFNT are critical for establishment and maintenance of pregnancy during the peri-implantation period of gestation in sheep.

Project description:Racemic difluoromethylornithine (D/L-DFMO) is an inhibitor of ODC (ornithine decarboxylase), the first enzyme in eukaryotic polyamine biosynthesis. D/L-DFMO is an effective anti-parasitic agent and inhibitor of mammalian cell growth and development. Purified human ODC-catalysed ornithine decarboxylation is highly stereospecific. However, both DFMO enantiomers suppressed ODC activity in a time- and concentration-dependent manner. ODC activity failed to recover after treatment with either L- or D-DFMO and dialysis to remove free inhibitor. The inhibitor dissociation constant (K(D)) values for the formation of enzyme-inhibitor complexes were 28.3+/-3.4, 1.3+/-0.3 and 2.2+/-0.4 microM respectively for D-, L- and D/L-DFMO. The differences in these K(D) values were statistically significant ( P <0.05). The inhibitor inactivation constants (K(inact)) for the irreversible step were 0.25+/-0.03, 0.15+/-0.03 and 0.15+/-0.03 min(-1) respectively for D-, L- and D/L-DFMO. These latter values were not statistically significantly different ( P >0.1). D-DFMO was a more potent inhibitor (IC50 approximately 7.5 microM) when compared with D-ornithine (IC50 approximately 1.5 mM) of ODC-catalysed L-ornithine decarboxylation. Treatment of human colon tumour-derived HCT116 cells with either L- or D-DFMO decreased the cellular polyamine contents in a concentration-dependent manner. These results show that both enantiomers of DFMO irreversibly inactivate ODC and suggest that this inactivation occurs by a common mechanism. Both enantiomers form enzyme-inhibitor complexes with ODC, but the probability of formation of these complexes is 20 times greater for L-DFMO when compared with D-DFMO. The rate of the irreversible reaction in ODC inactivation is similar for the L- and D-enantiomer. This unexpected similarity between DFMO enantiomers, in contrast with the high degree of stereospecificity of the substrate ornithine, appears to be due to the alpha-substituent of the inhibitor. The D-enantiomer may have advantages, such as decreased normal tissue toxicity, over L- or D/L-DFMO in some clinical applications.

Project description:We hypothesized that the enzyme arginase-1 is released into the cerebrospinal fluid (CSF) during red blood cell lysis and contributes to dysregulated metabolism of the nitric oxide (NO) precursor L-arginine during aneurysmal subarachnoid hemorrhage (SAH). This prospective case-control study included 43 patients with aneurysmal SAH and ventricular drainage for clinical reasons. Longitudinal CSF samples (99) were obtained in the course of SAH. Patients were dichotomized regarding the occurrence of cerebral vasospasm syndrome (CVS) (N = 19). Arginase-1 and the amino acids L-arginine and L-ornithine were quantified in CSF. Outcome assessments included delayed cerebral ischemia (DCI) and functional status after 3 months using the modified Rankin Scale (mRS). Arginase-1 was released into the CSF of SAH patients whereas this enzyme was undetectable in controls. Compared to patients without CVS, arginase-1 levels were higher in CVS patients until day 14 after clinical event. The well-known surrogate parameter for arginase acitivity, the L-arginine to L-ornithine ratio (Arg/Orn), correlated with CSF arginase-1 levels. Arg/Orn was reduced in patients with CVS from disease onset (days 1-3, p = 0.0009) until day 14. Logistic regression analysis of early Arg/Orn was predictive for CVS (p = 0.008) and DCI (p = 0.035), independent of age, Hunt and Hess grade, and intraventricular blood. Arg/Orn < 2.71 at disease onset predicted CVS with a sensitivity of 86.7% and specificity of 72.2%. Arg/Orn ≥ 2.71 predicted excellent functional outcome. We propose a novel mechanism contributing to NO deprivation during SAH: arginase-1 is released from erythrocytes into the CSF, leading to L-arginine consumption and reduced NO bioavailability. Furthermore, Arg/Orn is a robust predictor for occurrence of CVS, DCI, and functional outcome 3 months after aneurysmal SAH. Our data provide a novel prognostic biomarker and may contribute to the development of novel therapeutic strategies in SAH. Clinical Trial Registration-URL: http://www.drks.de . Unique identifier: DRKS00015293, date of registration: 13.09.2018.

Project description:Polyamines are important regulators of basal cellular functions but also subserve highly specific tasks in the mammalian brain. With this respect, polyamines and the synthesizing and degrading enzymes are clearly differentially distributed in neurons versus glial cells and also in different brain areas. The synthesis of the diamine putrescine may be driven via two different pathways. In the "classical" pathway urea and carbon dioxide are removed from arginine by arginase and ornithine decarboxylase. The alternative pathway, first removing carbon dioxide by arginine decarboxlyase and then urea by agmatinase, may serve the same purpose. Furthermore, the intermediate product of the alternative pathway, agmatine, is an endogenous ligand for imidazoline receptors and may serve as a neurotransmitter. In order to evaluate and compare the expression patterns of the two gate keeper enzymes arginase and arginine decarboxylase, we generated polyclonal, monospecific antibodies against arginase-1 and arginine decarboxylase. Using these tools, we immunocytochemically screened the rat brain and compared the expression patterns of both enzymes in several brain areas on the regional, cellular and subcellular level. In contrast to other enzymes of the polyamine pathway, arginine decarboxylase and arginase are both constitutively and widely expressed in rat brain neurons. In cerebral cortex and hippocampus, principal neurons and putative interneurons were clearly labeled for both enzymes. Labeling, however, was strikingly different in these neurons with respect to the subcellular localization of the enzymes. While with antibodies against arginine decarboxylase the immunosignal was distributed throughout the cytoplasm, arginase-like immunoreactivity was preferentially localized to Golgi stacks. Given the apparent congruence of arginase and arginine decarboxylase distribution with respect to certain cell populations, it seems likely that the synthesis of agmatine rather than putrescine may be the main purpose of the alternative pathway of polyamine synthesis, while the classical pathway supplies putrescine and spermidine/spermine in these neurons.

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DL-alpha-difluoromethyl[3,4-3H]arginine metabolism in tobacco and mammalian cells. Inhibition of ornithine decarboxylase activity after arginase-mediated hydrolysis of DL-alpha-difluoromethylarginine to DL-alpha-difluoromethylornithine.

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