Polyamine reutilization and turnover in brain

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  • Neurochemical Research, Vol. 10, No. 4, 1985


    NIKOLAUS SEILER AND FRANK N. BOLKENIUS Merrell-Dow Research Institute Strasbourg Center

    16 rue d'Ankara 67084 Strasbourg France

    Accepted November 19, 1984

    N~ ,N2-bis-(2,3-butadienyl)-1,4-butanediamine (MDL 72527) is an irreversible, spe- cific inhibitor of polyamine oxidase, which allows one to completely inactivate this enzyme in all organs of an experimental animal. As a result one observes a linear increase of N~-acetylspermidine and N~-acetylspermine concentrations in brain. The rate of accumulation seems directly proportional to the rate of sper- midine, and spermine degradation respectively, and since no compensatory changes of the polyamine synthetic enzymes were induced by inhibition of po- lyamine oxidase, the rate of acetyl-polyamine accumulation is assumed to be a measure for polyamine turnover. The decrease of brain putrescine levels by 70 percent in the brains of MDL 72527-treated animals suggests the quantitative sig- nificance of putrescine reutilisation. Pretreatment of the animals with D,L-e~-di- fluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase re- duced both, polyamine turnover rate and the extent of putrescine reutilization. Inhibition of GABA-T produced a significant increase of polyamine turnover in brain, in agreement with the known induction of ornithine decarboxylase activity after treatment with inhibitors of GABA-T.


    Polyamine oxidase (flavin dependent ; PAO) catalyses the degradation of N l -acety lspermid ine and N l -acety lspermine to putrescine and spermidine respect ively (1, 2). Together with acety lCoA:po lyamine Nl -acety l t rans - ferase (3) it const i tutes the enzymat ic machinery which is responsible for the intracel lular po lyamine catabol ism and turnover. As appears from Figure 1 inhibit ion of PAO should produce an accumulat ion of the N l- acetylder ivat ives of spermidine and spermine, and a concomitant de- crease of spermidine and putrescine.

    529 0364-3190/85/0400-0529504.50/0 9 1985 Plenum Publishing Corporation


    Recently Nl,N2-bis-(2,3-butadienyl)-l,4-butanediamine (MDL 72527) became available (4). This compound is an enzyme-activated, irreversible inhibitor of PAO, both in vitro (4) and in vivo (5), of high potency and specificity. Parenteral doses >20 mg per kg body weight produce a near to complete inactivation of PAO in all organs of mice. According to the expectations, Nl-acetylspermidine and Nl-acetylspermine concentrations were enhanced inall organs (5), including the brain. Since the compound showed no toxic effects, even after 6 weeks of treatment with doses, which produced a complete blockade of PAO, we assumed that the metabolic changes observed in an animal after inactivation of PAO reflect the phys- iological situation. Thus MDL 72527 is expected to become a powerful tool in the study of quantitative aspects of intracellular polyamine catab- olism.

    In the present work we are exploring the use of MDL 72527 for the determination of polyamine turnover rates and polyamine reutilization in mouse brain.


    Chemicals. Usual laboratory chemicals were from E. Merck (Darmstadt, Germany) or Baker Chemicals (Deventer, The Netherlands). N1,N12-Diacetylspermine, N~-acetylsper - mine and Nl-acetylspermidine were prepared as hydrochlorides according to published pro- cedures (2, 6). D,L-4-aminohex-5-enoic acid (~/-acetylenicGABA) (MDL 71645) and D,L-a- difluoromethylornithine (DFMO) (MDL 71782) are products of the Merrell Dow Research Institute, Strasbourg. N l,N~2-bis-(2,3-butadienyl)-1,4-butanediamine dihydrochloride (MDL 72527) was synthetised by P. Bey.

    Laboratory Animals. Male CD~ mice (weighing 30 to 35 g) from Charles River (St. Aubin- les-Elbeuf, France) were kept in groups of ten at standardized conditions (22~ 60% tel. humidity, 12 hr light, 12 hr dark cycle) and had free access to standard diet and water.

    Preparation of Brain Extracts. The animals were decapitated and brains were removed from the skull; heads inserted in ice-cold physiol, saline). If brain parts (hemispheres, brain stem, cerebellum, and medulla) were analysed these structures were dissected and pooled from four brains.

    For the determination of PAO-activity and polyamines, brains were homogenized with 4 vol. of borate buffer pH 9.0 (50 mM boric acid, 1 mM dithiothreitol, 1 mM EDTA).

    A portion of this homogenate was mixed with an equal volume of 0.4 M HC104 and the supernatant was used for polyamine determinations.

    For the determination of ornithine decarboxylase (ODC) and S-adenosyl-methionine de- carboxylase (SAMDC) activities brains were homogenised with 4 vol of 0.03 M sodium phosphate buffer pH 7.1 (0.1 mM EDTA, 0.1 mM pyridoxal-5'-phosphate, 5 mM dithioth- reitol).

    Enzyme Assays. Polyamine oxidase (PAO) activity was determined in brain homogenates, using N~,N~2-Diacetylspermine dihydrochloride as substrate (7). The activities of acetylCoA:polyamine N~-acetyltransferase (8) ornithine decarboxylase (ODC) (9) and S- adenosylmethionine decarboxylase (SAMDC) (10) were determined in the I00,000 g super-


    natants of homogenates in phosphate buffer, according to published procedures. In order to avoid any ~4CO2-formation from labelled ornithine by a reaction sequence which is started by transamination of ornithine, the mixtures for ornithine decarboxylase assay contained 1 mM D,L-4-aminohex-5-ynoic acid (11).

    Assay of Polyamines and Acetylpotyamines. Polyamines and their derivatives were de- termined by reversed phase HPLC of the ion pairs with octane sulfonate, and fluorimetric evaluation of the reaction products with o-phthalaldehyde/2-mercaptoethanol (12).


    Administration of a single intraperitoneal dose of 50 mg MDL 72527 per kg body weight produced a complete inactivation of brain PAO ac- tivity within less than 30 rain (results not shown). In contrast with pe- ripheral organs (5) N~-acetylspermidine concentration in brain increased linearly with time (increment 1. I nmol per g brain per hr) over three days. In Figure 2 the results for the first 24 hr after administration of the PAO inactivator are shown. Putrescine concentration decreased linearly during the first 8 hr at the same rate, as Nl-acetylspermidine concentration in- creased, and reached a minimum at about 24 hr. Even daily treatment for 7 days with the same dose of MDL 72527 did not decrease brain putrescine levels further.

    N~-Acetylspermine concentration also increased to well measurable concentrations within 24 hr at a rate of about 0.05 nmol per g brain per hr and spermidine levels decreased correspondingly.

    Groups of mice received for one week a 3 percent solution of the ODC inhibitor DFMO instead of drinking fluid. Their average drug intake cor- responded to about 4 g per kg body weight per day. As appears from Figure 2 these animals had considerably lower putrescine levels than con- trols, and their rate of Nl-acetylspermidine accumulation, after admin- istration of 50 mg MDL 72527 per kg body weight was less than 50 percent. Although Nl-acetylspermine accumulation was still detectable, the rate of increase was slowed considerably (0.03 nmol g/brain/hr). Brain sper- midine and spermine levels were slightly lowered by this treatment but the differences between DFMO-treated animals and saline-treated con- trols were not significant.

    The comparison of the rates of N'-acetylspermidine accumulation in four major brain parts, after inactivation of PAO did not reveal great differences. The steepest increase with 1.4 nmol per g tissue per hr was found in cerebellum, the slowest with 0.89 nmol per g per hr in the brain stem. Extensive inhibition of ODC by administration of DFMO reduced in all parts the rate of N~-acetylspermidine accumulation by about 50 percent (Figure 3), in agreement with the findings in whole brain.


    9 NS- ( 2-Carboxyethyl ) -sPe rmldlne SPERMINE I SDermlc acid

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    9 ................... N8-Acetyl spe rmidlne 9

    N_ACety lput rean lne


    Put rean lne : SPERMIDINE ~ Isoput~eanlne 1scram

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    FIG. 1. Scheme of polyamine metabolism. 1 = Ornithine decarboxylase (ODC) 2 = S-adenosylmethione decarboxylase (SAMDC) 3 = Spermidine synthase 4 = Spermine synthase 5 = AcetyICoA :polyamine NLacetyltransferase (cytoplasmic) 6 = Acety lCoA:spermidine NS-acetyltransferase (nuclear) 7 = Polyamine oxidase (PAO) 8 = Monoamine oxidase 9 = Diamine oxidase (or other copper-containing amine oxidases)

    10 = Ornithine:2-oxoacid aminotransferase 11 = 4-aminobutyrate: 2-oxoacid aminotransferase (GABA-T) 12 = Glutamate decarboxylase (GAD).

    Administration of a single dose of MDL 72527 alone decreased brain putrescine levels within 24 hr by about 70 percent (Figure 4). Treatment with the ODC inhibitor for one week produced about the same, or even a slightly greater depletion of brain putrescine levels. Animals which were pretreated with DFMO showed a further small depletion of brain putres-

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    FIG. 3. Rates of Nl-acetylspermidine accumulation in four parts of mouse brain after a single intraperitoneal injection of 50 mg N~,N2-bis-(2,3-butadienyl)-l,4-butanediamine dihydroch- loride (MDL 72527) per kg body weight. The effect of administration of a 3% solution of D,L*c~-difluoromethylornithine (MDL 71782: DFMO) for one week, instead of drinking water, (Average drug intake 4 g per kg per day). Mean values of 3 animals _+ SD.

    cine levels, when MDL 72527 was given in addition. Similar observations were made with brain spermidine levels (Figure 5). Since spermidine/ spermine-ratios are obtained at a higher precision than absolute values, these ratios have been compared. The decrease of brain spermidine level was small, but statistically significant in all brain parts.

    ODC, SAMDC and acetylCoA: spermidine Nl-acetyltransferase activ- ities were not changed measurably in the various parts of mouse brain 24 hr after administration of MDL 72527, nor had the treatment with DFMO a detectable effect on SAMDC or acetyltransferase activity.


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    oL Fro. 4. Putrescine content in four parts of mouse brain after a single intraperitoneal dose of 50 mg N1,N2-bis-(2,3-butadienyl)-l,4-butanediamine dihydrochloride (MDL 72527), or after one week of treatment with a 3 percent solution of D,L-a-difluoromethylornithine (DFMO), or a combined treatment with the two compounds.

    The animals were decapitated 24 hr after MDL 72527 administration. The DFMO-treated groups had access to this drug until sacrifice.

    The columns represent the mean values from a total of 12 animals per treatment group _+ SD, i.e. four brain parts were pooled for polyamine (and enzyme) assays, and three pools were analysed separately. The asterisks indicate a statistically significant difference (P - 0.01) between treated and control group. (Student's t-test (two tailed)).

    Groups of mice received either a single intraperitoneal dose of the GABA-T inhibitors 4-aminohex-5-enoic acid (-y-vinylGABA; 750 mg/kg) (16) or 4-aminohex-5-ynoic acid (-y-acetylenic GABA; 75 mg/kg) (17) or they received these same doses on 9 consecut ive days. Twenty four hr after the last dose of the GABA-T inhibitors, 50 mg MDL 72527 per kg body weight was administered intraperitoneal ly and the brain po lyamine patterns were observed for the fol lowing 8 hr. Figure 6 summarizes the results of these exper iments. The observed enhancement of whole brain






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    legend to Fig. 4. An asterisk indicates a statistically significant difference (P -< 0.05) between treated and control group. Student's t-test (two tailed)).

    putrescine levels was m agreement with previously reported findings (18). Spermidine and spermine levels were not affected by treatment with the GABA-T inhibitors.

    Levels of Nl-acetylspermidine in the brain of GABA-T inhibitor treated mice were elevated even before MDL 72527 was administered. This in- crease was especially high in the animals which received -/-acetylenic GABA over 9 days. Inhibition of PAO produced a steep accumulation of Nl-acetylspermidine (and also of Nl-acetylspermine) in the GABA-T in- hibitor-treated mice. From the experimental data average values of ac- cumulation rates for Nl-acetylspermidine and Nl-acetylspermine were calculated (Table I). Putrescine levels in the GABA-T inhibitor-treated




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    FIG. 6~ The effect of treatment with GABA-T inhibitors on putrescine concentrations and the rate of Nl-acetylspermidine accumulation in mouse brain.

    Left panel (filled symbols) treatment with 3,-vinylGABA. Right panel (empty symbols) treatment with wacetylenicGABA.

    dots:single dose (750 mg -/-vinylGABA per kg body weight i.p.; 75 mg ~/-acetylenicGABA per kg body weight i.p.) diamonds:daily doses (same as above) for 9 days. triangles:control animals (treatment with physiol, saline).

    MDL 72527 was given 24 hr after the last dose of the GABA-T inhibitor. Each point is the mean value obtained from 3 or 5 animals; vertical bars indicate _+ SD.





    Treatment* N 1 _

    Acetylspermidine N~-Acetylspermine (nmol per g brain per h*)

    Physiological saline 1.11 ___ 0.02 (0.99) 0,051 _+ 0.008 (0,79) ~-VinylGABA 750 mg/kg, 1 dose 2.0 0.2 (0.91) 0.054 _+ 0.009 (0.79) 750 mg/kg per day, 9 days 2.4 0.2 (0.92) 0.09 -+ 0.01 (0.84) ~-AcetylenicGABA 75 nag/ 2.4 0.3 (0.89) 0.084 0.01 (0.81)

    kg, 1 dose 75 mg/kg per day, 9 days 3.4 -+ 0.4 (0.92) 0.113 _+ 0.009 (0.96)

    MDL 72527 was given 24 hr after the last dose of thee GABA-T inhibitor, and accumulation rates of Nl-acetylspermidine and Nl-acetylspermine were determined during the following 8 hr. * Statistical evaluation of the experimental data of Figure 6 by linear regression analysis :

    Mean values of the slope of the curves _+ SD ; r z in parentheses.

    animals were at too great variance, as to allow the detection of an influ- ence of PAO inhibition (Figure 6).


    Factors Affecting the Determination of Polyamine Turnover. Previous attempts to measure polyamine turnover rates were made by labelling the endogenous pools with suitable radioactive precursors and determination of the gradual decline of the specific radioactivity of spermidine and spermine. A critical assessment of this method has been published (19, 20). This type of approach is hampered by the interconversion of one polyamine into another. Furthermore it was shown that the observed half lives of the putrescine and aminopropyl moieties of spermidine and sperm- ine are apparently greatly different especially in brain. The calculated biological half life of spermidine and spermine was about 42 days, if la- belled putrescine was used as precursor, but it was only 13-16 days, if 3H- or 14C-labeled methionine was used for labeling the brain polyamines. In order to explain these findings it was suggested that a certain proportion of the putrescine and spermidine, which is formed by degradation of spermine and spermidine, along the interconversion pathway (21) can be reutilized for the formation of spermidine and spermine, respectively.


    Figure 1 is a recent version of the originally suggested (19) scheme which demonstrates the reactions involved in polyamine interconversion, reu- tilization and catabolism.

    One molecule of methionine is needed for the substitution of each ami- nopropyl moiety, which is eliminated in the form of 3-acetamidopropanal during the cleavage of the Nl-acetylderivatives of spermidine and sperm- ine by PAO. In contrast, the putrescine moiety has to be substituted by decarboxylation of ornithine only to that extent, to which it is lost by degradation to GABA, excretion, or by degradation or elimination of sper- midine, spermine and their derivatives. Putreanine is a normal constituent of brain (22), but the rate of its formation seems to be slow (23), and the activity of diamine oxidase is very low in the mature vertebrate brains (24).

    It is not known presently to which extent polyamines are eliminated from brain via circulation or CSF. We conclude from the fact that N l- acetylspermidine concentration was linearly accumulating in brain for three days, from non-detectable levels to a final concentration of 60 nmol/ g (5) that elimination of this compound is slow, as compared with its formation.

    If the premises are correct, i.e. if indeed the degradation of the po- lyamines along pathways which are catalyzed by copper-containing amine oxidases are slow in vivo, and if elimination by transport is slow, the rate of Nl-acetylspermidine accumulation is a direct measure of spermidine turnover, because neither endogenous polyamine pools, nor the activity of the enzymes involved in polyamine metabolism were significantly in- fluenced by treatment with the PAO inhibitor. From the observed ac- cumulation rate of Nl-acetylspermidine and a measured spermidine pool of 410 nmol per g mouse brain one may calculate a biological half life of brain spermidine of 8 days. Taking a spermine pool of 310 nmol/g, and the rate of NJ-acetylspermine accumulation of 0.05 nmol/g/h (Table I) T89 for spermine appears to be 130 days.

    The -r89 for spermidine, as obtained from the measuremenent of N ~- acetylspermidine accumulation is smaller than that obtained previously (13-16 days) from experiments with labelled methionine (19). However, it should be noted that the method of inhibiting PAO demonstrates pref- erentially the fastest pools, whereas after extended administration of the radioactive precursor, slow endogenous polyamine pools are also la- belled. Moreover, methionine is incorporated into, and gradually released from proteins. Thus it will be incorporated into polyamines not only during the actual labelling period, but also during the period when the decline of the specific radioactivity is determined. This causes an apparent pro- longation of the experimentally determined biological half life.


    A greater problem is the explanation of the very serious discrepancy between the previously determined ,r I of 18 days for spermine, using labelled methionine as precursor (19) and the present value, as calculated from N~-acetylspermine accumulation rates (~'~ ~ 130 days). It is known that N~-acetylspermine is accumulating in tissues in situations of en- hanced polyamine degradation (8, 25), but it does not appear in the urine, even when both, PAO and copper-containing amine oxidases were inhib- ited (26). In contrast with N~-acetylspermidine, N~-acetylspermine has an unsubstituted aminopropyl-moiety, which may permit a metabolic route that is not relevant for Nl-acetylspermidine. For example, plasma amine oxidase attacks exclusively the aminopropyl-, not the aminobutyl- moiety of spermidine (27). In principle, Nl-acetylspermine could also serve as substrate of the acetylCOA:polyamine acetyltransferase. The product of this reaction, Nl,N~Z-diacetylspermine would escape obser- vation. Thus, owing to its structural characteristics, N~-acetylspermine could be metabolized and eliminated by an unknown pathway. If this was true for brain the observed accumulation rate of N~-acetylspermine would not reflect the actual rate of spermine degradation. Furthermore, if sperm- ine itself is significantly catabolized along pathways other than by inter- conversion, its biological half life would be much shorter than one as- sumes from the rate of accumulation of N~-acetylspermine.

    Inhibition of Ornithine Decarboxylase Activity and Polyamine Turn- over. From the decrease of putrescine levels by about 70 percent after inhibition of PAO, one can conclude that in brain about 70 percent of the putrescine is formed by spermidine degradation, and only about 30 per- cent originates directly from the decarboxylation of ornithine. The ex- tensive reutilization of putrescine for polyamine biosynthesis, which has been postulated previously (19, 20), is thus obvious. Polyamine reutiliz- ation is considered to be part of the regulation of cellular polyamine levels in highly differentiated, non proliferating cells (19, 20).

    Administration of DFMO depletes not only the proportion of putres- cine, which is deriving directly from decarboxylation of ornithine, but it also affects the putrescine, which is formed by degradation of spermidine, as appears from a simple analysis of the data summarized in Figure 4. The obvious reason for this effect is the reduction of the spermidine turn- over rate by about 50 percent. The reduction of spermidine turnover im- plies not only a reduced rate of putrescine reutilization, but most probably also a significant reduction of putrescine catabolism and elimination from the brain. This explains the small increment in the decline of brain pu- trescine levels, as compared with the rate of N~-acetylspermidine accu- mulation in DFMO-treated mice (Figure 2).


    The increased polyamine turnover in the brains of mice which were treated with the GABA-T inhibitors -y-vinylGABA and ~-acetylenicGABA was indicated by the elevated levels of Nl-acetylspermidine, and has been demonstrated by the increased rate of Nl-acetylspermidine accumulation after inhibition of PAO (Figure 6).

    Inhibition of GABA-T and Polyamine Degradation. It has previously been shown that treatment with -/-vinylGABA produces a rapid dose- dependent decrease of SAMDC-activity in brain, which is later followed by the elevation of ODC activity and by elevated brain putrescine levels (18). As appears from the present work, brain polyamine metabolism is greatly enhanced both by treatment with ~/-vinylGABA, but especially by -/-acetylenic GABA. The latter compound is not only a potent inhibitor of GABA-T, but it also inhibits GAD (17) and ornithine aminotransferase (28).

    Although it was not possible to measure an enhancement of acetylCoA:polyamine N~-acetyltransferase activity in the brains of GABA-T inhibitor-treated mice, the observed increase of N~-acetylsper - midine accumulation rate may nevertheless be explained on the basis of an enhanced acetylation rate. The method of measurement of acetyltrans- ferase activity is not sensitive enough, as to allow one to obtain precise values in brain homogenate (limit of sensitivity about 10 nmol/g/h).

    Pajunen et al. (29) reported that administration of allylglycine, a well known indirect inhibitor of GAD, also induces the decrease of SAMDC activity. At the same time a decrease of GABA, spermidine, and spermine levels is observed. In a recent report, it was shown that allylglycine ad- ministration produced a rapid increase of spermidine acetylation (30). This, together with the before mentioned results suggests an enhancement of polyamine degradation in brains, after GAD inhibition by allylglycine. In the case of the GABA-T inhibitor, this effect seems to be compensated by an increased rate of putrescine formation. Putrescine is not only a substrate, but also an activator of SAMDC (31), so that elevated putres- cine levels may compensate for the decreased SAMDC activity in the brains of the GABA-T inhibitor treated animals, so that as a net result, polyamine turnover is enhanced.

    What are the Reasons for the Absence of Physiological Consequences of PAO Inhibition? As was described in the Results part, complete in- activation of PAO in the brain is followed by a significant decrease of putrescine and an increase of Nl-acetylspermidine levels, with a small effect only on spermidine concentrations.

    It is not clear at present why these biochemical changes seem not to have obvious physiological consequences, even in an organ, such as the brain, with a highly regulated homeostasis.


    If the major function of spermidine is the optimization of protein bio- synthesis the decrease of brain spermidine concentration achievable by inhibition of PAO is presumably not sufficiently great as to result in ob- vious distrubances of protein biosynthesis (see 32).

    The pharmacology of Nl-acetylspermidine has not yet been studied; toxic effects have been observed after i.p. doses >1 g (33), however, these have not been correlated with brain levels of N~-acetylspermidine. The present state of the investigations resembles the time when inhibitors of MAO were administered without knowing their actual biological ef- fects: No behavioral changes were observed in this situation in experi- mental animals, inspite of profund changes of brain amine levels, as turned out later.

    Only careful pharmacological investigations of the PAO inhibitors and of the compounds which are affected by these drugs will presumably clarify this puzzling situation.


    Complete inactivation of brain PAO, using a single dose of N1,N2-bis - (2,3-butadienyl)-l,4-butanediamine (MDL 72527) and measurement of rates of Nl-acetylspermidine seems a simple, well reproducible method for the determination of spermidine interconversion rates in brain, and in brain structures. The method is restricted only by the fact that it is presently not known, whether and to what extent the observed accu- mulation rates are maximal, i.e. it is not clear, whether substantial amounts of endogenously formed Nl-acetylspermidine are removed from the brain as such, or after metabolism, via circulation or CSF. If this was the case the biological half lives which were calculated from the experimental data are expected to be too long.


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