DFMO Research Articles

Inhibition of polyamine metabolism in Phytophthora species

The effects of inhibitors of polyamine metabolism on the growth, ornithine decarboxylase (ODC) and polyamine concentration of Phytophthora infestans have been investigated, and comparisons made with Phytophthora cactorum and Phytophthora nicotianae . The enzyme-activated inhibitor of ODC, difluoromethylornithine (DFMO) at 0·1 m m inhibited the radial growth of P. infestans after 21 d to ca 30% of the water control. Using a homologous series of diamines, (NH 2 (CH 2 ) x NH 2 . 2HCl), full reversal of growth inhibition due to DFMO was achieved when x = 4, 5 and 10; and no reversal was found when x = 3 or 7. Spermidine, spermine and agmatine also fully reversed the inhibition due to DFMO. The inhibitor of arginine decarboxylase, monofluoromethyldehydroarginine, was almost as effective as DFMO as a growth inhibitor. The growth of P. cactorum and P. nicotianae was relatively insensitive to DFMO. The in vitro activity of the ODC was reduced by 0·1 m m DFMO, monofluoromethyldehydroomithine or methylacetylenic putrescine to less than 3% and 5% of the control in P. infestans and P. cactorum , respectively. In P. infestans (grown with or without 1 m m DFMO), putrescine, spermidine or spermine were all less than 10 nmol g −1 fresh weight. However, in P. cactorum , spermidine was present at 1·4 mol g −1 fresh weight, and was reduced to 38% by 1 m m DFMO. The results suggest that the greater growth inhibition in P. infestans caused by the inhibitors of polyamine metabolism may be related to the inherently smaller polyamine concentration, which probably results from the relatively low ODC activity found in this fungus.

Interactions of polyamines and nitrogen nutrition in plants

Biogenic amines occupy an important position among the many nitrogenous plant compounds. Polyamines are part of the overall metabolism of nitrogenous compounds, yet they do not seem to function in the ‘normal’ nitrogen nutrition. Rather, these widespread polycations (e. g. putrescine, spermidine and spermine) are involved in the regulation of growth and stress, probably by binding to negatively charged macromolecules. In addition, some diamines and polyamines are metabolized to yield 'secondary ’metabolites such as nicotine and other alkaloids. Previous studies have indicated that the ratio of nitrate to ammonium nutrition affects polyamine biosynthesis and content in intact plants. Thus, an increase in putrescine accumulation was found under conditions of excess ammonium ions, relative to nitrate. Modifications of nitrogen sources in the culture medium of tobacco cell suspensions (depletion of ammonium nitrate, or potassium nitrate, or both) resulted in marked changes in the content of cellular free polyamines. Considerable changes in the content of specific polyamines were also found with exposure to specific inhibitors of polyamine biosynthesis (difluoromethyl ornithine, difluoromethyl arginine, cyclohexylamine, methylglyoxal‐bis‐guanylhydrazone). However, a combination of nitrogen depletion of the medium and some inhibitors resulted in a very marked over‐production of spermidine and spermine. The significance of these findings is discussed in relation to the assumption that polyamines act as a metabolic buffer, and maintain cellular pH under conditions where ammonium assimilation produces an excess of protons.

Glucocorticoid and polyamine involvement in zinc uptake by COMMA-1D mammary epithelial cells.

The objective was to determine if a mammary cell line shows glucocorticoid stimulation of Zn uptake, and to determine whether polyamines mediate this stimulation. 65Zn uptake by COMMA-1D mouse mammary epithelial cells over a 24-h period increased significantly in cells administered 10(-7) or 10(-6) M hydrocortisone. Incorporation of 65Zn over a 1-h period was not hydrocortisone-responsive, suggesting that these incubation times represent uptake into different pools. The rate of entry into the cells over a 15-min period was significantly increased by supplementing cells with hydrocortisone with or without prolactin. Initially, cells grown in lactogenic hormone-supplemented media (10(-6) M hydrocortisone + 5 micrograms/mL ovine prolactin) had up to 65% greater 65Zn uptake over 24 h than cells in nonsupplemented growth media. 65Zn uptake from hormone media with the spermidine synthesis inhibitor methylglyoxal-bis-(guanylhydrazone) (MGBG, 10(-5)M) added was less than from growth media. Exogenous spermidine (10(-6)-10(-3)M) added to the MGBG + hormone media increased 65Zn uptake. Difluoromethylornithine (DFMO), an inhibitor of spermidine synthesis that blocks ornithine decarboxylase, caused a slight dose-dependent decrease in 65Zn uptake over the range 10(-6)-5 x 10(-3)M (p < 0.002) and tended to decrease 65Zn-uptake in lactogenic hormone-stimulated cells with 8 h of incubation, but not at other times. These data show that Zn uptake in mammary epithelial cells can be hormonally mediated by glucocorticoids and suggest that polyamines may be intracellular mediators of this effect.

Energetics of nutrition and polyamine-related tumour growth alterations in experimental cancer

The aim of this study was to evaluate whether food intake modulates experimental tumour growth by acute alterations in the energy state and blood flow of the tumour, and if so whether such changes are related to alterations in the enzyme ornithinedecarboxylase (ODC) and DNA synthesis. Inbred mice (C57BL/J) bearing a syngeneic undifferentiated and rapidly growing tumour were used. The tumour levels of high energy phosphates were measured in vivo by 31-P-NMR spectroscopy and biochemically following tissue extraction. DNA synthesis was estimated by measuring the incorporation of bromodeoxy-uridine into tumour DNA. Difluoro-methylornithine (DFMO) was used to inhibit ODC-activity. Tumour blood flow was estimated by a 132Xe local clearance technique. Tumour progression was associated with a significant decrease in tumour tissue high energy phosphates. Acute starvation decreased DNA-synthesis and tumour energy charge as well as its PCr/Pi which were rapidly normalised during subsequent refeeding. These changes were related to similar alterations in tumour blood flow. The inorganic phosphate (Pi) resonance and the resonances in the phosphomonoester (PME) region were considerably increased in tumour tissue. Inhibition of ODC-activity by DFMO decreased DNA-synthesis, which was associated with a secondary increase in tumour high energy phosphates probably due to a lowered energy demand for tumour cell division. The results demonstrate that host undernutrition was translated into retarded tumour growth associated with a decrease in the energy state and blood flow of the tumour. The results have bearing for the evaluation and planning of all treatment protocols with potential influence on food intake in experimental tumour-bearing animals.

Ototoxicity of Chemotherapeutic Agents

This chapter summarizes the reported ototoxicity data on the most clinically important ototoxic chemotherapeutic agents, notably emphasizing the oto(neuro)toxicities of the more commonly administered platinum compounds, cisplatin and carboplatin. Currently, in the United States, the only other marketed ototoxic chemotherapeutic agents are nitrogen mustard, alpha-difluoromethyl ornithine (DFMO), and the vinca alkaloids (vincristine and vinblastine sulfate); for these groups, animal ototoxicity data is sparse, and audiovestibular records of human ototoxicity are not available from most prospective, randomized controlled clinical trials. Future phase I, II, and III clinical oncologic trials of "experimental" chemotherapeutic agents should include methodology for audiovestibular monitoring, just as present FDA-approved cancer protocols with either monotherapy or combined therapy of known "ototoxic" agents should include standardized audiovestibular assessment in the database. Finally, continued clinical application of cisplatin alone or in combination with other chemotherapeutic agents in the successful treatment of solid tumors mandates decreasing or eliminating specifically the dose-dependent sensorineural hearing loss (partially in cases of complete or long-term partial remission) in addition to other common antiproliferation-induced side effects (nephritis, peripheral neuropathy, intractable nausea and vomiting, electrolyte imbalance, anaphylactic-like reactions, and myelosuppression). Because of the chemotherapeutic superiority of cisplatin, it is essential to continue to investigate methods of altering the dose-limiting oto(neuro)toxicity without causing a "counterproductive" reduction of the antitumor activity of cisplatin (or other second- or third-generation ototoxic platinum agents).

Enhanced stimulus-secretion coupling in polyamine-depleted rat insulinoma cells. An effect involving increased cytoplasmic Ca2+, inositol phosphate generation, and phorbol ester sensitivity.

To extend previous observations on the role of polyamines in insulin production, metabolism, and replication of insulin-secreting pancreatic beta cells, we have studied the role of polyamines in the regulation of the stimulus-secretion coupling of clonal rat insulinoma cells (RINm5F). For this purpose, RINm5F cells were partially depleted in their polyamine contents by use of the specific ornithine decarboxylase inhibitor difluoromethylornithine (DFMO), which led to an increase in cellular insulin and ATP contents. Analysis of different parts of the signal transduction pathway revealed that insulin secretion and the increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) after K(+)-induced depolarization were markedly enhanced in DFMO-treated cells. These effects were paralleled by increased voltage-activated Ca2+ currents, as judged by whole-cell patch-clamp analysis, probably reflecting increased channel activity rather than elevated number of channels per cell. DFMO treatment also rendered phospholipase C in these cells more sensitive to the muscarinic receptor agonist carbamylcholine, as evidenced by enhanced generation of inositol phosphates, increase in [Ca2+]i and insulin secretion, despite an unaltered ligand binding to muscarinic receptors and lack of effect on protein kinase C activity. In addition, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate, at concentrations suggested to be specific for protein kinase C activation, evoked an increased insulin output in polyamine-deprived cells compared to control cells. The stimulatory effects of glucose or the cyclic AMP raising agent theophylline on insulin release were not increased by DFMO treatment. In spite of increased binding of sulfonylurea in DFMO-treated cells, there was no secretory response or altered increase in [Ca2+]i in response to the drug in these cells. It is concluded that partial polyamine depletion sensitizes the stimulus-secretion coupling at multiple levels in the insulinoma cells, including increased voltage-dependent Ca2+ influx and enhanced responsiveness to activators of phospholipase C and protein kinase C. In their entirety, our present results indicate that the behavior of the stimulus-secretion coupling of polyamine-depleted RINm5F insulinoma cells changes towards that of native beta cells, thus improving the usefulness of this cell line for studies of beta cell insulin secretion.

The Effect of Ornithine Decarboxylase Inhibition on Growth, Enzyme Activities, and Polyamine Concentrations in Crinipellis perniciosa

Growth, polyamine concentrations, and the activities of enzymes of polyamine biosynthesis and breakdown were examined in the agaric Crinipellis perniciosa (Stahel) Sing., the causal agent of witches′-broom disease of cocoa (Theobroma cacao L.), that had been grown in the presence of the following irreversible inhibitors of ornithine decarboxylase: difluoromethylornithine (DFMO), monofluoromethyl-dehydro-ornithine methyl ester (MFMOCH3), and 6-heptyne 2,5 diamine (RRMAP), and in the presence of difluoromethylarginine (DFMA), an irreversible inhibitor of arginine decarboxylase. All treatments reduced mycelial growth and depressed ornithine decarboxylase activity, but these events were not always directly linked to polyamine depletion. Putrescine concentration was markedly increased by DFMO but was not affected by DFMA and was decreased by both RRMAP and MFMOCH3. All treatments, apart from MFMOCH3, increased spermidine. No spermine was detectable in DFMO- and MFMOCH3-treated mycelia. All treatments, apart from DFMA, increased S-adenosylmethionine decarboxylase activity. The activities of di- and polyamine oxidase were increased by DFMO but not significantly affected by other treatments. It is suggested that the growth reduction induced by DFMO, DEMA, and RRMAP may be due, in part, to spermidine accumulation. In this scheme, the action of polyamine oxidase on spermidine would generate hydrogen peroxide and free radicals, both of which would induce membrane damage.

Polyamines are essential for cell transformation by pp60v-src: delineation of molecular events relevant for the transformed phenotype.

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, becomes upregulated during cell proliferation and transformation. Here we show that intact ODC activity is needed for the acquisition of a transformed phenotype in rat 2R cells infected with a temperature-sensitive mutant of Rous sarcoma virus. Addition of the ODC inhibitor alpha-difluoromethyl ornithine (DFMO) to the cells (in polyamine-free medium) before shift to permissive temperature prevented the depolymerization of filamentous actin and morphological transformation. Polyamine supplementation restored the transforming potential of pp60v-src. DFMO did not interfere with the expression of pp60v-src or its in vitro tyrosine kinase activity. The tyrosine phosphorylation of most cellular proteins, including ras GAP, did not either display clear temperature- or DFMO-sensitive changes. A marked increase was, however, observed in the tyrosine phosphorylation of phosphatidylinositol 3-kinase and proteins of 33 and 36 kD upon the temperature shift, and these hyperphosphorylations were partially inhibited by DFMO. A DFMO-sensitive increase was also found in the total phosphorylation of calpactins I and II. The well-documented association of GAP with the phosphotyrosine-containing proteins p190 and p62 did not correlate with transformation, but a novel 42-kD tyrosine phosphorylated protein was complexed with GAP in a polyamine- and transformation-dependent manner. Further, tyrosine phosphorylated proteins of 130, 80/85, and 36 kD were found to coimmunoprecipitate with pp60v-src in a transformation-related manner. Altogether, this model offers a tool for sorting out the protein phosphorylations and associations critical for the transformed phenotype triggered by pp60v-src, and implicates a pivotal role for polyamines in cell transformation.

Alterations in repair of alkylating agent-induced DNA damage in polyamine-depleted human cells

Treatment of HeLa cells with the polyamine biosynthesis inhibitors difluoromethylornithine (DFMO) and/or methylglyoxal bis(guanylhydrazone) (MGBG) results in marked depression in levels of the cellular polyamines putrescine, spermidine and spermine. Cells in this polyamine-depleted state exhibited increased sensitivity to monofunctional alkylating agents, manifested as decreased cloning ability and retardation of the DNA excision repair process. DFMO treatment did not alter the initial level of interaction of radiolabeled alkylating agent with cellular DNA, but combined treatment with DFMO and MGBG reduced covalent binding, probably through effects on cell cycling. Polyamine supplementation had no effects on initial yield of DNA single-strand breaks in drug-treated cells. The repair defect appeared similar to that observed previously in polyamine-depleted cells following X-irradiation and UV irradiation, namely retarded sealing of DNA strand breaks. It was not possible to reverse the effects of these inhibitors by short periods of polyamine loading, despite the fact that all three polyamines could be restored to near-normal levels. These findings provide the first demonstration of altered response of polyamine-depleted cells to monofunctional alkylating agents and contribute to our understanding of altered responses of polyamine-depleted cancer cells to a variety of DNA-reactive chemotherapeutic drugs.

Human promyelocytic cell line HL60 has the specific binding sites for prolactin and its ornithine decarboxylase, DNA synthesis and cellular proliferation are induced by prolactin

Human prolactin (hPRL) induced ornithine decarboxylase (ODC) activity, subsequently DNA synthesis and cellular proliferation on human promyelocytic cells, HL60, cultured in a serum-free medium. HL60 cells had 2100 specific binding sites for hPRL per cell, showing a dissociation constant of 1.1 × 10 −10 M. Binding of 125I-PRL to the cells was not blocked by simultaneous addition of human growth hormone. ODC activity and DNA synthesis were activated maximally at 5 and 20 h, respectively, after the addition of 0.05 nM hPRL. These effects of PRL on cellular proliferation, ODC activity and DNA synthesis were abolished by the simultaneous addition of anti-hPRL antibody. Simultaneous addition of an irreversible inhibitor of ODC, difluoromethyl ornithine (DFMO), also abolished the inductions of ODC and DNA synthesis by hPRL. The inhibitory effect of DFMO on hPRL-induced DNA synthesis was reversed by the addition of putrescine to the culture medium. These results suggest that hPRL binds to the prolactin receptor on HL60 cells and induces ODC activity to increase cellular polyamine levels, which eventually stimulates DNA synthesis and cellular proliferation.

Inhibitory effect of EGF on secretory response of rat parietal cells is associated with an induction of ODC

The present studies were designed to evaluate the parietal cell acid production in response to short-time stimulation by epidermal growth factor (EGF). Studies were performed in vitro using isolated cells from rat stomachs, and acid production was indirectly determined by [14C]aminopyrine accumulation. EGF inhibited histamine-stimulated aminopyrine accumulation from standard incubation medium (K+ = 5 mM) but not from that with increased K+ concentration (K+ = 70 mM). EGF significantly stimulated ornithine decarboxylase (ODC) activity, an effect that was blocked by the specific ODC inhibitor, difluoromethylornithine (DFMO). In the presence of DFMO, EGF failed to inhibit histamine-stimulated aminopyrine uptake. Like EGF, the polyamine spermine, which is a direct product of enhanced ODC activity, also inhibited histamine-stimulated aminopyrine uptake. Unlike EGF, the spermine-induced inhibition of aminopyrine accumulation was not altered by DFMO. Thus the DFMO effect was specific to EGF. Taken together, these results are consistent with the postulate that EGF inhibits parietal cell secretory response through the induction of ODC activity and increased synthesis of polyamines.

Alpha 1-antitrypsin as a biomarker in azoxymethane induced intestinal tumors in F344 rats

Alpha 1-antitrypsin ( α 1AT) has been detected in several human tumors and is thought to be a marker of neoplastic change and progression. As the biology of azoxymethane (AOM) induced intestinal tumors in F344 rats has many characteristics of human intestinal tumors, we have investigated α 1AT expression in AOM induced rat intestinal tumors. Nine of 12 colonic carcinomas and 6 8 small intestinal carcinomas had α 1AT positive tumor cells. Only 1 11 colonic adenomas and 0 3 small intestinal adenomas contained α 1AT. Thus α 1AT is a marker of malignancy in this model. Inhibition of carcinogenesis by piroxicam and difluoromethyl ornithine inhibited α 1AT expression.

Effect of polyamine biosynthesis inhibitors on mycelial growth and concentrations of polyamines in Ophiostoma ulmi (Buism.) Nannf.

Several inhibitors of polyamine biosynthesis reduced mycelial growth of the fungus Ophiostoma ulmi (Buism.) Nannf. cultured on malt extract-agar medium. Growth inhibition was particularly evident when expressed in terms of fresh weight rather than colony diameter. Of the different drugs tested, the most effective was difluoromethylarginine (DFMA), while difluoromethylornithine (DFMO) and methylglyoxal(bis)guanyl-hydrazone (MGBG) plus cyclohexylamine (CHA) reduced growth only by 75 and 62% respectively. The growth inhibition by DFMO + DFMA, measured as colony diameter, was apparently reversed by putrescine (PUT), but not when expressed in terms of fresh weight. Spermidine (SPD) was the most abundant polyamine in control cultures, followed by PUT and spermine (SPM). PUT was no longer detectable 8 d after inoculation. On day 10, DFMO and DFMA, alone and in combination, caused a significant reduction in cellular SPD concentrations, while exogenously supplied PUT restored the levels of this polyamine to control values. MGBG + CHA caused a conspicuous accumulation of PUT and an approximately 50% reduction in SPD titres. DFMA, alone or in combination with DFMO and with or without PUT, led to increased cellular levels of SPM. The latter polyamine, but not PUT or SPD, strongly retarded growth when added to the growth medium. As suggested by the effectiveness of DFMA in inhibiting growth, arginine decarboxylase activity was shown to be prevalent over ornithine decarboxylase activity in this fungus.

Effects of gastrin and difluoromethylornithine on growth of human colon cancer.

The effect of difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase activity, was evaluated in vivo and in vitro on the growth of a gastrin-sensitive human colon carcinoma (WiDr). In vivo, mice bearing the tumor treated with pentagastrin had larger tumors with higher ornithine decarboxylase activity and polyamine content (P < 0.05) than mice not treated with pentagastrin. Difluoromethylornithine treatment significantly decreased ornithine decarboxylase in both the pentagastrin-treated and the untreated animals; however, DFMO had no effect on tumor volume, weight, protein, or DNA content. In cell culture, gastrin treatment increased WiDr cell number and [3H]thymidine incorporation in the presence or absence of serum. In serum-free conditions, however, gastrin stimulated cell growth without concomitantly increasing ODC activity. DFMO, on the other hand, decreased both ODC activity and growth. These studies suggest that the trophic effect of gastrin on WiDr human colon cancer is independent of ODC activity. Since gastrin treatment increased ODC activity in vivo, gastrin may interact in vitro with other factors present in serum that can alter ODC activity.

Studies on the effects of an ornithine decarboxylase inhibitor on lupus nephritis reveal a post-transcriptional modification of the enzyme.

Upregulation of ornithine decarboxylase (ODC) activity and polyamine levels is found in the kidney of MRL-lpr/lpr (lpr) mouse, an animal model of lupus. To understand the molecular genetics of ODC regulation in lpr mouse, we analyzed ODC mRNA and activity in the kidney of lpr and normal BALB/c and MRL(-)+/+ mice. Although ODC activity was significantly higher in lpr kidney, its mRNA level was lower compared to normal strains, as measured by Northern blot hybridization. Treatment of lpr mouse with difluoromethylornithine (DFMO) reduced ODC activity in lpr kidney to the level of normal strains. In contrast, ODC mRNA level increased 12-fold by DFMO treatment. These results suggest that post-transcriptional modification of ODC in lpr genetic background might be responsible for increased ODC activity and polyamines. The beneficial effect of DFMO on murine lupus suggests a pathogenic role for altered ODC regulation in lpr mouse.

Ammonium requirement for radiation‐induced accumulation of polyamines in suspension‐cultured grape cells

The effects of radiation‐mediated free radical production on polyamine metabolism were investigated in grape cells (Vitis vinifera L. cv. Gamay) using a cell suspension culture. Putrescine (Put) synthesis was triggered in irradiated cells (0. 5 kGy) only when ammonium was present in the culture medium. Under these conditions. Put accumulated to a continuously high level. As also described for other kinds of stress, the level of spermidine was slightly enhanced and that of spermine unchanged. The role of ammonium was assessed by studying non‐irradiated cell cultures. In the presence of ammonium, a transient increase of both arginine decarboxylase (ADC) activity and of Put synthesis was observed during the lag phase of growth. This Put enhancement was inhibited by difluoromethyl arginine and not by difluoromethyl ornithine, showing that increased Put synthesis occurs via the ADC pathway. When ammonium was withheld from the culture medium. ADC activity was still triggered though transient Put accumulation was completely suppressed. These results emphasize the importance of ammonium availability in cultured cells as a limiting factor for Put production. Polyamine synthesis, therefore, cannot be stimulated by gamma irradiation in the absence of an ammonium supply. These results support the hypothesis that Put synthesis is a detoxification process of the ammonium produced as a result of nitrogen recycling within stressed plant cells.

Polyamines and Intestinal Epithelial Hyperplasia in Streptozotocin-Diabetic Rats

We measured specific activity of ornithine decarboxylase (ODC) and contents of putrescine and of the polyamines (spermidine and spermine) in isolated villus and crypt enterocytes from the jejunum of adolescent streptozotocin-diabetic and weight-matched control rats and diabetic and control rats treated with difluoromethyl ornithine (DFMO) 10 days after induction of diabetes. Consistent with previous observations by others of elevated ODC activity and contents of putrescine and of the polyamines in the intestinal epithelium undergoing hyperplasia, our studies showed elevated ODC activity and contents of putrescine and spermidine, but not of spermine, in the hyperplastic intestinal epithelium of diabetic rats. As in previous studies, suppression of ODC activity by DFMO prevented not only the jejunal epithelial hyperplasia in the diabetic rats, but also retarded jejunal epithelial growth in the control rats. DFMO administration lowered ODC activity by over 80% in both diabetic and control rat enterocytes and prevented the rise in enterocyte contents of putrescine and spermidine in the diabetic rat. The observation that, in both diabetic and control rats, treatment with DFMO lowered spermidine content in the crypt enterocytes but had no similar consistent effect on contents of putrescine or spermine suggested that spermidine could have been responsible for the intestinal epithelial hyperplasia in the diabetic rats and for the normal growth of the intestinal epithelium in control rats.

Effect of difluoromethylornithine on atrial natriuretic peptide in rat atria

Polyamines are aliphatic cations known to have a role in cellular growth and differentiation. In this study, difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis, was used to investigate its effect on atrial natriuretic peptide (ANP) in atria of rat. The rats were given DFMO (2%) in drinking water for 10 days and three intraperitoneal administrations (200 mg/kg), 24, 18 and 1 h prior to experiment. Radioimmunoassay of ANP in atrial extracts indicated that DFMO treatment increased ANP contents of atria. ANP from atrial extracts was immunoprecipitated using ANP antibody and the immunoprecipitate was resolved and detected by SDS-PAGE and Western blotting. The predominant ANP peptide in both control and DFMO group migrated at 17 kDa. The synthesis of ANP was studied following the intravenous administration of [ 35S]methionine. ANP in atrial extracts was immunoprecipitated by ANP antibody. The incorporation of ANP into control and DFMO group peaked at 30 min and returned to a basal level by 60 min. DFMO decreased the incorporation of [ 35S]methionine into ANP. At 30 min following the administration of [ 35S]methionine, putrescine restored the synthesis of tricholoroprecipitable atrial proteins, but had no effect on the synthesis of ANP. At 60 min following, the amount of labeled ANP in DFMO + putrescine-treated group was significantly lower than that in DFMO group. These results indicate that polyamines influence both the synthesis and secretion of ANP.

The effect of difluoromethylornithine on polyamine levels in pollinated and napthaleneacetic acid-induced young tomato fruits

The polyamine synthesis inhibitor difluoromethylornithine (DFMO) inhibits growth and cell division in tomato fruits (Lycopersicon esculentum Mill. c.v. F 121) when applied after pollination by the split-stem technique. In napthaleneacetic acid (NAA)-induced fruits, however, growth is not inhibited by DFMO. We analyzed the effect of DFMO on cell division in auxin induced tomato fruits, and on the concentration of free, soluble conjugated and insoluble bound polyamines in pollinated and NAA-induced fruits, five days after pollination in the presence and absence of DFMO. Cell number was not significantly reduced by DFMO in NAA-induced fruits five days after fruit set. Free spermine, soluble conjugated and insoluble bound polyamines were higher in fruits treated with DFMO than in those treated with water. They were also higher in DFMO-treated pollinated fruits than in NAA-induced ones. Our results suggest that inhibition of cell division by polyamine synthesis inhibitor might affect further metabolism of polyamines.

Inhibition of renal ornithine decarboxylase activity prevents kidney hypertrophy in experimental diabetes.

The selective ornithine decarboxylase (ODC) inhibitor difluoromethyl ornithine (DFMO) was used to investigate the role of polyamines in initial diabetic renal enlargement. ODC activity in kidneys from diabetic animals was increased (fivefold) 24 h after diabetes induction (P < 0.05), and throughout the study (7 days) the activity remained 2- to 3-fold elevated (P < 0.05). Insulin treatment normalized renal ODC activity, whereas DFMO treatment totally inhibited the kidney ODC activity. The kidney weight in diabetic rats was 21% higher than that of control rats (1,074 +/- 35 mg and 889 +/- 16 mg, P < 0.001). Insulin treatment normalized kidney weight (847 +/- 13 mg). Despite unaltered diabetic metabolic aberrations the kidney weight in DFMO-treated diabetic rats was normalized (911 +/- 7 mg). In conclusion, the ODC activity in diabetic kidneys undergoing hypertrophy was increased. Insulin treatment normalized both kidney weight and kidney ODC activity. Finally, selective inhibition of ODC activity by DFMO resulted in kidneys of normal size, despite unaltered diabetic metabolic aberrations. These findings support the hypothesis that polyamines play an important role in initial diabetic renal enlargement.