Małgorzata Manteuffel-Cymborowska

Agmatine modulates the in vivo biosynthesis and interconversion of polyamines and cell proliferation.

Agmatine has recently gained wide interest as a bioactive arginine metabolite with a multitude of physiological functions. This study evaluates the in vivo role of agmatine in the modulation of metabolism and intracellular level of polyamines. Here, we report that agmatine, administered to mice, differentially affects the renal and liver activity of the two key enzymes regulating polyamine biosynthesis and interconversion/degradation. Thus, agmatine exerts a negative regulation of ODC activity and protein content, and positive regulation of SSAT activity, having no effect on ODC and SSAT transcript level. Agmatine modulation of ODC and SSAT activities is noticeably augmented by the inhibitor of its catabolism, aminoguanidine. Antizyme and eIF4E protein content appears to be affected by agmatine only insignificantly and apparently do not contribute to agmatine-induced down-regulation of ODC content. The homeostasis of spermidine and spermine is preserved after agmatine injection, while the putrescine level decreases. Furthermore, when tested in a mouse kidney injury model, agmatine, partially but significantly, reduces [3H] thymidine incorporation into DNA. This is consistent with suppressed renal tubule epithelial cell proliferation. The findings provide in vivo evidence of a substantial role of agmatine as a modulator of polyamine biosynthesis and degradation and suggest its suppressive effect on cell proliferation.

Arginine and ornithine metabolizing enzymes in testosterone-induced hypertrophic mouse kidney

Administration of testosterone to female mice causes hypertrophy of their kidneys with spectacular induction of ornithine decarboxylase and significant increase in the level of putrescine. We tried to find out whether testosterone treatment affects also the renal activities of enzymes participating in the formation and utilization of ornithine, specifically arginase and ornithine aminotransferase, and whether they are dependent on putrescine level. Swiss, CFW, DBA2 or F1 (CFW x DBA2) female and male mice were injected with testosterone (125 mg/kg) or CB 3717 (100 mg/kg). DFMO was applied in the drinking water. The activities of the enzymes were determined 24 hr or 5 days after administration of CB 3717 or testosterone, respectively. Renal activities of ornithine decarboxylase (ODC), arginase and ornithine aminotransferase (OAT) were found to be sex-differentiated. The highest activity of ODC was characteristic for the kidneys of males, whereas those of arginase and OAT for the kidneys of females. In the kidneys of testosterone-treated female mice a decrease (50%) of OAT, and a significant increase of arginase activities (up to 200%), were observed. In the males these changes were less pronounced. DFMO, which completely inhibited the activity of renal ODC, did not influence significantly the testosterone-induced arginase and the testosterone-decreased OAT. Arginase and OAT, in contrast to ODC, were not changed in CB 3717-induced hyperplastic kidney. The study showed testosterone-induced differential changes in the activity of two enzymes involved in ornithine biosynthesis and catabolism which accompanied ODC induction in female mouse kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyamines in testosterone-induced hypertrophic and antifolate-induced hyperplastic mouse kidney. Differential effect of α-difluoromethylornithine

In the testosterone-induced hypertrophic and antifolate (N10-propargyl,5,6-dideazafolic acid, CB 3717)-induced hyperplastic mouse kidney models, a marked increase of two diamine levels--putrescine and cadaverine--occurred which paralleled induced ornithine decarboxylase (ODC) activity. Under these conditions the augmentation of spermidine levels was much smaller, while spermine levels were affected differentially--increased by testosterone and decreased by CB 3717; this resulted in an increase of spermidine/spermine ratio in hyperplastic, but not hypertrophic kidney. alpha-Difluoromethylornithine (DFMO) prevented testosterone- or CB 3717-induced increment of both diamine levels. Spermidine and spermine depletion in response to DFMO was significant in hyperplastic kidney only. DFMO also significantly affected the other biochemical markers of hyperplasia, namely lowered CB 3717-induced cell proliferation rate and increased S-adenosylmethionine decarboxylase (AdoMetDC) activity. In contrast, testosterone-induced hypertrophy was not influenced by DFMO, as judged by the lack of its effect on S-adenosylmethionine synthetase and cystathionine and synthase activity. These results indicate that the increase of putrescine levels does not mediate testosterone-induced renal hypertrophy and possibly also antifolate-induced hyperplasia. The involvement of spermidine in mediation of renal hyperplasia is highly possible, while that of spermine is excluded.

Catecholamines are required for androgen-induced ODC expression but not for hypertrophy of mouse kidney.

Catecholamine depletion, evoked by reserpine, dramatically impaired (5-fold) the testosterone-induced increase of ornithine decarboxylase (ODC) activity in female mouse kidney. However, reserpine did not prevent kidney hypertrophy evoked by testosterone. This is evidenced by the activity of sensitive, biochemical markers of renal hypertrophy, namely arginase and ornithine aminotransferase (OAT), that responded with the increase and decrease of activities to testosterone treatment, respectively. Arginine and ornithine, substrates and/or products of marker enzymes, showed a striking homeostasis as their level was not affected by testosterone and reserpine, and only slightly by DFMO. Northern blot analysis revealed that the ODC mRNA level, that was increased 10-fold by testosterone, was decreased 2-fold in catecholamine-depleted hypertrophic kidney. Thus, ODC transcript level, lowered by reserpine, correlated partially with an attenuated response of ODC activity to testosterone. This was in contrast to DFMO, which inhibited ODC activity, but significantly increased its mRNA content. It is concluded that catecholamines could be involved together with testosterone in regulation of the ODC gene expression in mouse kidney.

The interaction of folate analogues with dihydrofolate reductase from Galleria mellonella

Abstract The interaction of aminopterin and some folate and pretidine analogues with dihydrofolate reductase from Galleria mellonella has been investigated. Differences in their mode of action dependent on the pH of the reaction mixture have been found. The inhibition screening of several analogues either of the folate molecule or its pteridine fragment revealed that the 4-amino analogues of folate were the most effective inhibitors of this enzyme.

Cross-talk between steroid-receptor-mediated and cell-membrane-receptor-mediated signalling pathways results in the in vivo modulation of c-Met and ornithine decarboxylase gene expression in mouse kidney

The cross-talk in vivo between two signalling pathways activated by testosterone via intracellular androgen receptor, and induced by damage to renal tubules evoked by anti-folate [N(10)-propargyl-5,8-dideazafolic acid (CB 3717)] or folate is reported. We show that CB 3717/folate induces the expression of the hepatocyte growth factor (HGF)/c-Met signalling system in injured kidneys in which a significant, but transient, elevation of the HGF mRNA level occurs. It is followed by a severalfold increase in the c-Met transmembrane receptor message that persists for up to 24 h. The c-Met expression is also positively controlled by testosterone, which induces a significant increase in its mRNA level that is abolished by an anti-androgen, casodex. However, when testosterone and anti-folate/folate are administered sequentially, a substantial (3.5-4.0-fold) decrease in the increase of c-Met expression caused by CB 3717/folate alone occurs. Similarly, testosterone-induced ornithine decarboxylase (ODC) mRNA level and activity are decreased 2.8-7.7-fold when the androgen is applied together with CB 3717. Antagonism between these pathways is also visible under physiological conditions in the kidneys of male mice in which, owing to elevated endogenous testosterone levels, neither the ODC activity nor the mRNA level is induced by anti-folate/folate, whereas the c-Met message response to these drugs is significantly decreased. Our results document a substantial negative regulation of c-Met and ODC gene expression as a result of the cross-talk between testosterone-activated and HGF-activated pathways and suggest a sex-differentiated response to injury of mouse kidneys.

Variability in response of dihydrofolate reductase from Galleria mellonella towards urea, urea-like compounds and salts

Abstract 1. 1. The dihydrofolate reductase m the post-mitochondrial supernatant from Galleria mellonella fat body was found to be activated by urea, urea-like compounds and salts. 2. 2. The urea activated enzyme as well as guanidine-HCl or salt activated enzyme exhibited only one p H optimum in contrast to the two p H optima,characteristic of the non-activated enzyme. 3. 3. Dihydrofolate reductase partially purified on Sephadex G-100 was also activated by guanidine-HCl and NH 4 C1, but only immediately after dution, when it exhibited an activity profile with two p H optima. 4. 4. During storage the enzyme spontaneously underwent an activation correlated with the changes in its p H. activity profile. Such an enzyme was inhibited by guanidine-HCL and NH 4 Cl.

Ornithine decarboxylase induction in mouse kidney as indicator of renal damage. Differential nephrotoxic effect of anticancer antifolate drugs

High doses of folate and its quinazoline analogue with antitumour activity, N10-propargyl-5,8-dideazafolic acid (CB 3717), caused severe renal damage in mice, leading in the case of folate to death. The mouse kidneys increased in weight, which was accompanied by time- and dose-dependent induction of ornithine decarboxylase (ODC) activity. In contrast, methotrexate (MTX) had negligible effect on mouse kidneys except when applied together with the non-steroidal antiinflammatory drug, indomethacin.

Catecholamines participate in the induction of ornithine decarboxylase gene expression in normal and hyperplastic mouse kidney

In the quinazoline antifolate (CB 3717)-induced hyperplastic kidney model, a remarkable increase of ornithine decarboxylase (ODC) activity was paralleled by a smaller, but highly significant augmentation of the ODC transcript level. Catecholamine depletion, evoked by reserpine, strongly impaired antifolate-induced ODC expression; the enzyme activity was almost completely abolished while the mRNA level decreased by 60%. Moreover, under conditions of a depleted catecholamine pool, kidney enlargement was significantly reduced confirming our earlier reports on the indispensability of ODC induction for renal hyperplasia (M. Manteuffel-Cymborowska et al. , Biochim. Biophys. Acta, 1182 (1993) 133-141[1]). In normal mouse kidney catecholamines appeared to be inducers of ODC expression. Use of selective agonists of catecholamine receptors demonstrated the importance of dopamine D2 receptors, and to a lower extent beta adrenoreceptors, in the catecholamine mediation of induction of ODC activity and of ODC mRNA levels. These increases were not abolished by an antiandrogen, casodex, suggesting that catecholamine control of ODC expression is an androgen receptor-independent process. The results obtained point to the critical role of renal catecholamines; these biogenic amines are not only involved in the regulation of ODC expression in normal kidney but are also required for the induction of ODC in hyperplastic kidney evoked by antifolate and, as shown recently (M. Manteuffel-Cymborowska et al., Biochim. Biophys. Acta, 1356 (1997) 292-298[2]), in testosterone-induced hypertrophic kidney.

Participation of SH groups in the auto-activation of insect dihydrofolate reductase

Abstract 1. 1. Some extraneous proteins retarded the auto-activation of the insect dihydrofolate reductase. Their effect seems to be connected with protection of the enzyme SH groups. 2. 2. All tested thiol compounds prevented and reversed the auto-activation of dihydrofolate reductase. 3. 3. The enzyme was found to be activated by several SH reagents and their effect was also prevented and reversed by thiol compounds. 4. 4. The supposition was made that during the process of auto-activation of the insect dihydrofolate reductase, oxidation of its SH groups occurs, which results in a catalytically more favourable enzyme conformation.