Polyamine reutilization and turnover in brain

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

POLYAMINE REUTILIZATION AND TURNOVER IN BRAIN

N I K O L A U S S E I L E R AND F R A N K N . B O L K E N I U S 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, specific 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 spermidine, and spermine degradation respectively, and since no compensatory changes of the polyamine synthetic enzymes were induced by inhibition of polyamine 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~-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase reduced 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.

I N T R O D U C T I O N

P o l y a m i n e oxidase (f lavin d e p e n d e n t ; PAO) ca ta lyses the degrada t ion of

N l - a c e t y l s p e r m i d i n e and N l - a c e t y l s p e r m i n e to pu t resc ine and spermid ine respec t ive ly (1, 2). Toge the r with a c e t y l C o A : p o l y a m i n e N l - ace ty l t r ans - ferase (3) it cons t i tu t e s the e n z y m a t i c m a c h i n e r y which is r e spons ib le for the in t race l lu la r p o l y a m i n e ca t abo l i sm and tu rnover . As appears f rom Figure 1 inh ib i t ion of P A O should p roduce an a c c u m u l a t i o n of the N l- ace ty lde r iva t ives of spe rmid ine and spe rmine , and a c o n c o m i t a n t de- c rease of spe rmid ine and pu t resc ine .

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

530 SEILER AND BOLKENIUS

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 physiological situation. Thus MDL 72527 is expected to become a powerful tool in the study of quantitative aspects of intracellular polyamine catabolism.

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.

EXPERIMENTAL PROCEDURE

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 procedures (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 decarboxylase (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 dithiothreitol).

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-

POLYAMINE REUTILIZATION AND TURNOVER 531

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 determined by reversed phase HPLC of the ion pairs with octane sulfonate, and fluorimetric evaluation of the reaction products with o-phthalaldehyde/2-mercaptoethanol (12).

RESULTS

Administration of a single intraperitoneal dose of 50 mg MDL 72527 per kg body weight produced a complete inactivation of brain PAO activity 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 increased, 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 administration 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 spermidine 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.

532 S E I L E R A N D B O L K E N I U S

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10 = Orni th ine :2-oxoacid aminot ransferase 11 = 4-aminobutyra te : 2-oxoacid aminot ransferase (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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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 activities 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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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)).

G r o u p s of mice rece ived e i ther a single in t raper i tonea l dose of the G A B A - T inhib i tors 4 -aminohex-5 -eno ic acid (-y-vinylGABA; 750 mg/kg)

(16) or 4 - aminohex -5 -yno ic acid (-y-acetylenic G A B A ; 75 mg/kg) (17) or they rece ived these same doses on 9 consecu t ive days. T w e n t y four hr

af ter the last dose of the G A B A - T inhibi tors , 50 mg M D L 72527 per kg body weight was admin i s t e r ed in t raper i tonea l ly and the bra in p o l y a m i n e pa t t e rns were o b s e r v e d for the fo l lowing 8 hr. F igure 6 summar i ze s the resul ts of these expe r imen t s . The obse rved e n h a n c e m e n t of whole b ra in


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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 increase 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 inhibitor-treated mice. From the experimental data average values of accumulation rates for Nl-acetylspermidine and Nl-acetylspermine were calculated (Table I). Putrescine levels in the GABA-T inhibitor-treated

POLYAMINE REUTILIZATION AND TURNOVER

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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.


TABLE I RATES OF ACCUMULATION OF NI-AcETYLSPERMIDINE AND NI-AcETYLSPERMINE IN

THE BRAINS OF MICE FOLLOWING INTRAPERITONEAL ADMINISTRATION OF 50 MG

MDL 72527 PER KG BODY WEICHT

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

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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 influence of PAO inhibition (Figure 6).

D I S C U S S I O N

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 spermine are apparently greatly different especially in brain. The calculated biological half life of spermidine and spermine was about 42 days, if labelled 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, reutilization and catabolism.

One molecule of methionine is needed for the substitution of each aminopropyl moiety, which is eliminated in the form of 3-acetamidopropanal during the cleavage of the Nl-acetylderivatives of spermidine and spermine 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 spermidine, 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 polyamines 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 accumulation 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) T�89 for spermine appears to be 130 days.

The -r�89 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 labelled. 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 enhanced 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 spermine itself is significantly catabolized along pathways other than by interconversion, 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 percent originates directly from the decarboxylation of ornithine. The extensive reutilization of putrescine for polyamine biosynthesis, which has been postulated previously (19, 20), is thus obvious. Polyamine reutilization 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 putrescine, 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 turnover 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 putrescine levels, as compared with the rate of N~-acetylspermidine accumulation 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 acetyltransferase 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 administration 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 putrescine 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 o f Physiological Consequences of PAO Inhibition? As was described in the Results part, complete inactivation 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 biosynthesis the decrease of brain spermidine concentration achievable by inhibition of PAO is presumably not sufficiently great as to result in obvious 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 effects: No behavioral changes were observed in this situation in experimental 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.

CONCLUSION

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 accumulation 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.

REFERENCES

1. SELLER, N. 1981. Amide-bond-forming reactions of polyamines. Pages 127-149, in MOR- RIS, D. R., and MARTON, L. J. (eds.) Polyamines in biology and medicine. Marcel Dek- ker, New York, Basel.

2, BOLKENIUS, F. N., and SEILER, N. 1981. Acetylderivatives as intermediates in polyamine catabolism. Int. J. Biochem. 13:287-292.

3. PEGG, A. E., SEELY, J. E., P6sO, H., DELLA RAGIONE, F., and ZAGON, I. S. 1982. Polyamine biosynthesis and interconversion in rodent tissues. Fed. Proc, 41:3065-3072.

4. BOLKErqUS, F. N., BEY, P., and SEILER, N. 1984. Biochemical properties of some enzyme-activated irreversible inhibitors of polyamine oxidase (PAO). Abstr. No. 51. In- tern. Conf. on Polyamines, Budapest, Hungary, August 6-10.


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6. TABOR, H., TABOR, C. W., and DE MEIS, L. 1971. Chemical synthesis of N-acetyl-1,4- diaminobutane, NLacetylspermidine and NS-acetylspermidine. Meth. Enzymol. 17b:829-833.

7. SELLER, N., BOLKEmUS, F. N., KNODOEN, B., and MAMONT, P. 1980. Polyamine oxidase in rat tissues. Biochim. Biophys. Acta 615:480-488.

8. SELLER, N., BOLKENIUS, F. N., and SARHAN, S. 1981. Formation of acetypolyamines in the liver of fasting animals. Int. J. Biochem. 13:1205-1214.

9. ONO, M., INOUE, H., SuzuKI, F., and TAKEDA, Y. 1972. Studies on ornithine decarboxylase from the liver of thioacetamide-treated rats. Biochim. Biophys. Acta 284:285- 297.

10. PEoo, A. E., and W1LLIAMS-ASHMAN, H. G. 1969. On the role of S-adenosylmethionine in the biosynthesis of spermidine by rat prostate. J. Biol. Chem. 244:682-693.

11. SELLER, N., and SARHAN, S. 1980. On the nonoccurrence of ornitbine decarboxylase in nerve endings. Neurochem. Res. 5:97-100.

12. SELLER, N., and KNODOEN, B. 1980. High-performance liquid chromatographic procedure for the simultaneous determination of the natural polyamines and their monoacetyl derivatives. J. Chromatog. 221:227-235.

13. BEY, P. 1978. Substrate-induced irreversible inhibition of a-aminoacid decarboxylases. Application to glutamate, aromatic L-c~-aminoacid and ornithine decarboxylases. Pages 27-41, in SELLER, N., JUNG, M. J., and KOcH-WESER, J. (eds.) Enzyme-activated irreversible inhibitors. Elsevier, Amsterdam, New York, Oxford.

14. MAMONT, P. S., DUCHESNE, M.-C., JODER-OHLENBUSCH, A.-M., and GROVE, J. 1978. Effects of ornithine decarboxylase inhibitors on cultured cells. Pages 43-54, in SELLER, N., JUNG, M. J., and KOCH-WESER, J. (eds.) Enzyme-activated irreversible inhibitors. Elsevier, Amsterdam, New York, Oxford.

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16. JUNG, M. J., LIPPERT, B., METCALF, B. W., BOHLEN, P., and SCHECHTER~ P. J. 1977. 3,-VinylGABA (4-amino-hex-5-enoic acid), a new selective irreversible inhibitor of GABA-T: effects on brain GABA metabolism in mice. J. Neurochem. 29:797-802.

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26. SELLER, N., BOLKENIUS, F. N., and KN6D6EN B. 1985. The influence of catabolic reactions on polyamine excretion. Biochem. J. 225:219-226.

27. TABOR, C. W., TABOR, H., and BACHRACR, U. 1964. Identification of the aminoaldehydes produced by oxidation of spermine and spermidine in purified plasma amine oxidase. J. Biol. Chem. 239:2194-2203.

28. JUNG, M. J., and SELLER, N. 1978. Enzyme-activated irreversible inbibitors of L-ornithine:2-oxoacid aminotransferase. J. Biol. Chem. 253:7431-7439.

29. PAJUNEN, A. E. I., HIETALA, O. A., BARUCH-VIRRANSALO, E. L., and PIHA, S. S. 1979. The effect of O,L-allylglycine on polyamine and GABA metabolism in mouse brain. J. Neurochem. 32:1401-1408.

30. ORTIZ, J. G., GIACOBINI, E., and SCHMIDT-GLENEWINKEL, Z. 1984. Allylglycine affects acetylation of putrescine and spermidine in mouse brain. Neuropharmacol. 23:387-390.

31. WILLIAMS-ASHMAN, H. G., and PEGG, A. E. 1981. Aminopropyl group transfers in polyamine biosynthesis. Pages 43-73, in MORRIS, D. R., and MARTON, L. J. (eds.) Po- lyamines in biology and medicine. Marcel Dekker, New York, Basel.

32. RUDKIN, B. B., MAMONT, P. S., and SELLER, N. 1984. Decreased protein-synthetic activity is an early consequence of spermidine depletion in rat hepatoma tissue-culture cells. Biochem. J. 217:731-741.

33. BLANKENSHIP, J., and AL SHABANAH, O. A. 1983. Toxicology of Nl-acetylspermidine and NS-acetylspermidine in mice. Fed. Proc. 42; Abstr. No. 7625.

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Polyamine reutilization and turnover in brain