Developmental aspects of polyamine interconversion in rat brain

hat. J. Devl. Neuroscience. Vol. 4, No. 3, pp. 217-224. 1986.

0736-5748/86 $113.IXI+II.(K) Pergamon Journals Ltd. 1986 ISDN

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D E V E L O P M E N T A L ASPECTS OF P O L Y A M I N E I N T E R C O N V E R S I O N IN R A T BRAIN F. N. BOLKENIUSand N. SELLER Merrell Dow Research Institute, Strasbourg Center, 16. rue d'Ankara, 67084 Strasbourg Cedex, France (Accepted 31) November 1985)

Abstract--In the mammalian organism putrescine is formed by two reactions: (a) decarboxylation of ornithine and (b) degradation of spermidine via the so-called interconversion pathway. The latter comprises NLacetylation of spermidine by a cytosolic acetyltransferase, and oxidative splitting of NLacetylspermidine to putrescine by polyamine oxidase (PAO). It has previously been shown that specific inhibition of PAO causes a time-dependent accumulation of N Lacetylspermidine in brain, which is a measure of spermidine turnover. Another consequence of PAO inhibition is the decrease of brain putrescine concentration, proportional to its normal formation from spermidine. This observation allowed us to demonstrate the increasing significance of polyamine interconversion with brain maturation. The results support our hypothesis that the mechanisms which regulate cellular polyamine concentrations change during normal brain maturation from a system in which L-ornithine decarboxylase is dominating to a more sophisticated system in which both synthetic and catabolic processes become equally important regulatory factors. In contrast with current views, the activity of S-adenosylmethionine decarboxylase rather than that of ornithine decarboxylase limits the rate of polyamine biosynthesis during early brain development. In the mature brain the total amount of putrescine, which is formed both by decarboxylation of ornithine and by degradation of spermidine, limits the rate of spermidine formation. Changes of the regulatory system analogous to those described in this work are presumably not exclusive for brain, but rather characteristic for a variety of differentiating cells. Key words: NLAcetylspermidine, Polyamine oxidase, Polyamine turnover, Postnatal development,

Putrescine, Spermidine.

R e c e n t reviews o f metabolism and potential functions of the natural polyamines spermidine and spermine in vertebrate brain 19"2° reveal r e m a r k a b l e gaps in o u r knowledge and a low level of general interest in this area, despite the fact that p o l y a m i n e metabolism in brain seems to differ in several respects f r o m that in o t h e r organs. A m o n g the n u m e r o u s r e p o r t e d changes of e n z y m e and metabolite patterns during brain m a t u r a t i o n , the c o n c o m i t a n t decrease of ornithine decarboxylase ( O D C ; E C 4.1.1.17) activity I and putrescine c o n c e n t r a t i o n s 23 is most conspicuous. T h a t high putrescine c o n c e n t r a t i o n s are characteristic for i m m a t u r e brains and low concentrations for functionally m a t u r e brains is underlined by the fact that a u t o p h a g o u s in contrast with heterop h a g o u s vertebrates are delivered (hatched) with low brain putrescine concentrations. 24 T h e d e v e l o p m e n t a l pattern of S - a d e n o s y l m e t h i o n i n e decarboxylase ( S A M - D C , E C 4.1.1.50) activity is roughly inverse to that o f O D C 14 w h e r e a s the o t h e r e n z y m e s involved in p o l y a m i n e biosynthesis, spermidine and spermine synthase, exhibit only m o d e r a t e changes during brain d e v e l o p m e n t , iz Several years ago we p o s e d the question a b o u t the physiological significance o f the dramatic d r o p of putrescine levels during brain maturation. A first answer was o b t a i n e d when it was f o u n d that the putrescine m o i e t y o f spermidine and spermine had apparently a much greater biological half life than their a m i n o p r o p y l moieties. 2 F r o m this and o t h e r observations the conclusion was drawn that at least part of the putrescine which was f o r m e d by degradation of spermidine ("interconversion p a t h w a y " ) was re-used for spermidine synthesis. A n a l o g o u s l y the spermine-derived spermidine could be re-used ( " c o n c e p t of p o l y a m i n e re-utilization'). This implies that the aminopropyl moieties o f the p o l y a m i n e s are irreversibly eliminated from the metabolic cycle 2 (Fig. 1). In o t h e r words, p o l y a m i n e metabolism in the m a t u r e brain was a s s u m e d to be mainly achieved at the expense of m e t h i o n i n e , the p r e c u r s o r o f the a m i n o p r o p y l moieties, whereas the decarboxylation o f ornithine is required only to substitute for irreversible losses of putrescine, spermidine or s p e r m i n e by d e g r a d a t i o n o r elimination via circulation, TM o r for additional f o r m a t i o n of polyamines, if elevated c o n c e n t r a t i o n s were required. D~4.'3-~

217

218

F.N. Bolkenius and N. Scilcr

I0 SPERMINE

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~

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\

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lo

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Nl_Acetylspermidine /

2

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/

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Spermic acid

NI-Acetylsp ermlne

2

5'-Methylthio-4 CO2

~

Decarboxyl. SAM Acetate

N8-Acetylsp ermidlne 4 N_Acetylputreanine

~

N8_(2_Carboxyethyl)_spermldlne ~

7

l,o

~ CO

Glutamate1 2-Oxoglutarate

N-Acetyllsoput~eanlne

6

9NI-AcetylputPesclne~/ / "

~'2

X

Succlnlc ~ semlaldehyde

Fig. 1. Synthetic and catabolic reactions of polyamine metabolism in vertebrate organism. I. Ornithine decarboxylase (ODC). 2. S-Adenosylmethioninedecarboxylase (SAM-DC). 3. Spermidine synthase. 4. Spermine synthase. 5. AcetylCoA: spermidine NLacetyltransferase. 6. AcetylCoA: spermidine N~-acetyltransferase.7. NS-Acetylspermidinedeacetylase. 8. Polyamineoxidase (PAO). 9. Monoamine oxidase. 10. Copper amine oxidases. 11. Ornithine: 2-oxoglutarate aminotransferase. 12. 4-Aminobutyrate: 2-oxoglutarate aminotransferase. 13. Glutamate decarboxylase. The reactions involved in polyamine interconversion were subsequently clarified. 9"3" Figure 1 summarizes the most important reactions of polyamine metabolism. It appears from this figure that specific inhibition of polyamine oxidase (PAO), the enzyme splitting NLacetylspermidine to putrescine and NLacetylspermine to spermidine, 4''7 can be expected to result in the accumulation of the two substrates at a rate which equals the rate of spermine and spermidine degradation, respectively, if this process occurs in a closed system. With N',N4-bis(2,3-butadienyl)-l,4 butanediamine dihydrochloride ( M D L 72527), a specific and potent irreversible inhibitor of PAO, 3 these expectations proved to be correct. 5"27With this compound we had a tool suitable to determine (minimal) turnover rates of spermidine in vertebrate brains 2' and to obtain quantitative data on the extent of polyamine re-utilization in intact animals. TM In our present work we used N',N4-bis(2,3-butadienyl)-l,4-butanediamine.2HCl for the study of developmental aspects of polyamine interconversion in rat brain. Our data are in agreement with the notion that putrescine re-utilization is of increasing significance with brain maturation. EXPERIMENTAL PROCEDURES Materials Usual laboratory chemicals including o-phthalaldehyde were from Merck (Darmstadt, Germany). 1-Octane sulfonate was a product of Eastman Kodak (Rochester, NY). Putrescine dihydrochloride, spermidine phosphate and spermine phosphate were from Fluka (Buchs, Switzerland). AcetylCoA (Li salt) was from Boehringer (Mannheim, Germany). Dansyl chloride,'6 N'-acetylspermidine dihydrochloride,32 N',N'2-diacetylspermine dihydrochloride 4

219

Polyamine interconversion in rat brain and N I,N4-bis(2,3-butadienyl)-l,4-butanediamine dihydrochloride synthesized in our institute according to published methods.

(MDL

72527) 3

were

Animals Adult Sprague-Dawley and Buffalo rats were from Charles River (St. Aubin-les-EIbeuf, France). Rats were mated and the number of young per litter reduced where necessary to eight. Each litter was housed in a separate cage and at time intervals young were removed for sampling, always taking the same number of animals from each cage so that the litter sizes were equivalent. The young rats remained with their mothers until day 25 and were then separated according to sex and housed in groups of six. The housing conditions were standardized (22°C, 60% relative humidity; 12 hr light, 12 hr dark cycle; free access to standard diet and water). Only males were used in the case of adult rats.

Assay of polyamines Brains were removed from the skull of the decapitated animals and immediately frozen in liquid nitrogen. The frozen brains were homogenized with 10 vol. of ice-cold 0.2 N perchloric acid. After centrifugation the perchloric acid extracts were stored at 2°C until analysis. Polyamines were determined after separation of their ion pairs with 1-octane sulfonate on a reversed phase column by reaction with o-phthalaldehyde-2-mercaptoethanol-reagent, and recording of fluorescence intensity at 455 nm (excitation wavelength 345 nm). 22

Enzymic assays Polyamine oxidase (PAO). P A O was assayed in brain and liver homogenates, using Nt,N 12diacetylspermine as substrate. NLAcetylspermidine formation was determined by reaction with dansyl chloride and thin-layer chromatographic separation of the reaction products. 29 AcetylCoA: spermidine N I-acetyltransferase (cytosolic). This enzyme was determined using a previously published procedure. 31

DNA and protein D N A was separated from R N A and proteins using a modified Schmidt-Thannhauser procedure. 25 Deoxyribose served as standard. For protein determinations a modified Lowry procedure was used. 6

Calculations Statistically significant differences between groups were calculated by single factor analysis of variance. Multiple comparisons were done using Dunnet's t-test. 33

RESULTS

Polyamine oxidase activity during postnatal development Shortly after birth, P A O activity is low, both in brain and liver of Sprague-Dawley rats (Fig. 2). While its activity in liver increases roughly proportionately with the increase in weight, a very conspicuous increase of P A O activity is observed during the phase of slow brain growth between days 30 and 70 of postnatal life. After about 2 months the total PAO activity is in liver about 10 times higher than in brain. Very similar P A O activities were observed in brains of Buffalo rats. The data in Table 1 are expressed both as brain weight, and in D N A units (mg deoxyribose). The latter indicate that the average cellular P A O activity in brains of 6-month-old rats is about 6 times higher than on the first postnatal day. Changes in brain polyamine concentrations as a function of age and brain weight are shown in Table 2.

Rate of N I-acetylspermidine accumulation and decrease of brain putrescine concentration as a result of inhibition of polyamine oxidase Based on our experience with adult mice 21 an experimental protocol was followed which is described in the legend of Table 3. The amount of M D L 72527 given (50 mg/kg body wt) was

220

F . N . B o l k e n i u s and N. Seller

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Fig. 2. Developmental changes of polyamine oxidase activity in rat brain and liver. Each point represents the mean _+S.D. of five Sprague-Dawley rats. Table I. Activity of polyamine oxidase (PAO) in brains of Buffalo rats during postnatal development PAO-activity Age (days) 1 5 10 15 20 32 180

Brain weight (mg)

p,mol r.p.f./hr/ g brain

28(1 + 20 540 -+ 30 960+-40 1160-+50 1320-+211 1450-+40 1810-+70

0.66 _+(I.06 0.83 -+ 0.09 1.0 -+0.2 1.52-+0.07 * 1.3 _+(I.2" 1.4 -+0.3* 2.3 _+0.6*

/,tmol r.p.f./hr/ brain 0.19 0.48 1.0 1.8 1.8 2.2 4.2

ixmol r.p.f./hr/ mg deoxyribose

-+ (/.I13 -+ 0.(13" _+0.2* -+0.2* -+0.3* _+0.5* _+1.0"

1.5 .+ 0.2 2.6 -+ 0.5 3.1_+0.6 * 4.8+_0.3 * 4.1.+0.7" 4.8_+0.8 * 9.6_+2.7*

r . p . f . = r e a c t i o n product (NLacetylspermidine) formed from NI,NI2-diacetylspermine. Mean values -+S.D. ( N = 4 ) . * A statistically significant difference (P<~ 0.01) between PAO activity of l-day-old rats and older animals. Table 2. Concentration of putrescine, spermidine and spermine in the brain of Buffalo rats during postnatal development Concentration

Age (days)

Brain weight (mg)

Putrescine

Spermidine (p, mol/g brain)

Spermine

Spermidine plus spermine (p, mol per brain)

I 5 10 15 20 32 18(1

280 -+ 20 540 -+ 30 960-+40 1160 __50 1320 -+ 20 1450 _+40 1810 _+70

0.16 -+ 0.01 0.11 + II.(XI4 (I.(M6 + (I.(WI1 0.03 + 0.003 0.022 ± 0.0(12 0.010 ± 0.001 0.006 .+ 0.002

0.53 -+ 0.04 0.42 -+ 0.(XI5 0.4(l + 0.(11 (I.43 + 0.(13 (I.47 -+ 0.01 0.49 ± 0.04 I).47 -+ 0.01

1).40 -+ 0.02 0.32 _+(I.(XI4 0.36_+0.01 0.40 + 0.02 0.41 -+0.01 0.34 -+ 0.004 (L23 -+ I).(12

0.26 -+ 0.02 0.4(} +_0.005 0.73±0.01 0.96 -+ 0.06 1.16 +- 0.03 1.20 -+ 0.06 1.27 -+ (I.(15

Mean values ± S . D . ( N = 4 ) . NLAcetylspermidine concentration was below the detection limit of the method.

Polyamine interconversion in rat brain

221

sufficient to decrease P A O activity in brain and peripheral organs to levels below the detection limit of the enzyme assay, and it produced an increase of N~-acetylspermidine concentration in brain from non-measurable levels to very considerable values. From the NI-acetylspermidine concentration measured 24 hr after administration of the P A O inhibitor an average accumulation rate was calculated (Table 3). Similarly the percent decrease of putrescine was calculated from putrescine determinations in the brains of P A O inhibitor-treated and saline-treated (control) rats (Table 3). Table 3. The rate of N t-acetylspermidine accumulation and the relative decrease of putrescine concentration in brains of polyamine oxidase inhibitor-treated rats as a function of age

Age (days) 1 5 10 15 20 32 180

N '-Acetylspermidine accumulation rate (nmol/g brain/hr)

Percent decrease of putrescine concentration

0.54 ± 0.05* 0.48 -- 0.05* 0.82+-0.05 1.4 ±-0.1" 1.9 - 0 . 1 " 1.0 +-0.07

b.d.I. b.d.I. 13± 4* 46--- 6* 71± 2 60±11

0.8 ±1).2

7 8 -+

6

b . d . l . = b e l o w detection limit of the analytical method. Groups of 4 Buffalo rats received single s.c. doses of 50 mg MDL 72527 per kg body weight. Polyamine concentrations were determined 24 hr later, and compared with values obtained from saline-treated controls (mean values ± S.D.). An asterisk indicates a stastically significant difference ( P < 0 . 0 1 ) between the values of developing animals and adult (6-month-old) rats.

Administration of M D L 72527 also caused an increase of NI-acetyispermine concentration; however, it was too small to allow a meaningful interpretation. Our attempts to assay the activity of the cytosolic acetyltransferase, which is responsible for the formation of NI-acetylspermidine and Nl-acetylspermine were not successful because the activity of this enzyme never exceeded the sensitivity limit of the assay. 31 This is of the order of 1 nmol reaction product formed per 15 min, owing to a high background, which is due to the non-enzymic acetylation of the substrate by acetylCoA. The N~-acetylspermidine accumulation rates in Table 3 represent minimal values of spermidine degradation rates. The actual rates may be higher, because brain is not an ideally closed system. One has to assume that a certain portion of the endogenously formed Nt-acetylpolyamines is eliminated from the brain via circulation and CSF. T o assess the permeability of the blood-brain barrier for NI-acetylspermidine, a large dose of N~-acetylspermidine dihydrochloride was administered to 1-, 15- and 32-day-old rats, and N Iacetylspermidine concentrations were determined in blood and brain. Table 4 summarizes the experimental data. It appears that N~-acetylspermidine is only poorly entering the brain. The brain:blood ratios decreased somewhat with maturation. It is evident from the data in Table 4 that Nt-acetylspermidine is eliminated from the circulation much more rapidly in mature than in immature rats. One reason for this is most probably the great enhancement of P A O activity in liver, and possibly other organs, during growth. Although a precise comparison of N~-acetylspermidine accumulation rates in rats of different age is hampered by the concomitant decrease in the permeability of the blood-brain barrier, the following conclusions can nevertheless be drawn from the results in Table 3: (a) Polyamine interconversion is a physiological event even on the first day of postnatal life. (b) Spermidine degradation is enhanced between days 5 and 10. Maximum rates are attained around day 20. Thereafter one observes a gradual decline to adult values. (c) Inactivation of P A O in brain had no measurable effect on brain putrescine levels during the first 5 days of postnatal life. Thereafter

222

F.N. Bolkenius and N. Seller Table 4. Accumulation of parenterally administered N~-acetylspermidine in rat brain in relation to age N LAcetylspermidine concentration (nmol/g) Age (days)

Whole blood

Brain

Brain: blood ratio

1 15 32

19(X)± I(X) I(XIO_+ 2(X)* 9 0 _+ 40*

123 _+7 49 + 9* 1.6 + _1.0 ~

().064_+(k(~07 0.049_+ O.(X)2 0.019_+0.013 *

Groups of Buffalo rats received 51X) mg/kg (I.9 mmol/ kg) NI-acetylspermidine dihydrochloride s.c. Two hours later blood and brain samples were taken and analyzed by HPLC. -~2 Brain concentrations of NI-acetylspermidine were corrected for residual blood. 7"~ Mean values ± S . D . ( N - 3 ) . * A stastitically significant difference (P ~ 0.01 ) between I-day-old and older rats.

a gradually increasing relative difference between brain putrescine concentrations of animals with active (Table 2) and inactive PAO was observed until day 20, with no significant changes at later phases of development. The deficit of brain putrescine in PAO inhibitor-treated rats is proportional to the amount of putrescine which is normally formed by degradation of spermidine. DISCUSSION That spermidine is degraded at a significant rate in brains even of 1-day-old rats (Table 3) is not surprising in the light of the fact that at this time the main proliferative phase of neurons has terminated. The first 10 days of postnatal life of rats are mainly characterized by cell growth.~5 Moreover, even rapidly proliferating tumor cells, whose enzymatic machinery is geared to maximum rates of synthesis, show measurable rates of spermidine and spermine degradation along the interconversion path. 8"27 From the comparison of Tables 1 and 3 it seems that the activity of PAO as measured in vivo exceeds the physiological requirements. The PAO activity present in brain at a given time after birth would under favorable conditions (pH optimum, enzyme saturated with substrate) be sufficient to catabolize within 1 hr (days 1-5) or less ( > 10 days) Nt-acetylated polyamines in amounts equivalent to the total brain content of spermidine and spermine (Table 2). However, there are observations which indicate that under physiological conditions the PAO is not capable of completely oxidizing acetylpolyamines when these are formed at enhanced rates. For example, in fasted animals both NLacetylspermidine and N~-acetylspermine concentrations are increased in liver, and the former compound is excreted at an enhanced rate, although the PAO activity in liver is only slightly decreased. 3~ One may assume that at least in liver (and probably also in other organs) the accessibility of the substrates to PAO is limiting the reaction rate, owing to its localization in peroxisomes. Furthermore, both substrate concentration and pH are presumably suboptimal. Thus, the excessive developmental accumulation of PAO activity may be physiologically necessary to produce the observed increase in polyamine interconversion. However, an intact PAO activity is not of vital importance: even its complete inactivation for several weeks and the ensuing increased brain levels of NLacetylspermidine seem not to have any obvious toxic or behavioral effects. 5 The basis of P A O inhibition as a method for the determination of spermidine turnover rates in vertebrate brains has previously been established, and the limitations of the method have been discussed, zl If the spermidine pool does not change during the time of experimentation, the observed (average) NLacetylspermidine accumulation rates will equal minimal spermidine turnover rates. The assumption of a constant spermidine pool is in first approximation valid in our case, as can be seen from the data of Table 2. It appears (Table 3) that spermidine turnover is increasing during the first 20 days of postnatal life, and decreases thereafter to adult values.

Polyamine interconversion in rat brain

223

The development of the maximum of the turnover rate can qualitatively be explained as follows: in the brains of 1-10-day-old rats, the activity of SAM-DC is below the detection limit of the method. 14,26In contrast ODC activity is still high; 1"26on day 10 it exceeds with 1.4 - 0.3 nmol 14CO2 formed/g brain/hr 26 the observed NI-acetylspermidine accumulation rate. Thus it can be concluded that the (low) SAM-DC activity is limiting the rate of spermidine formation before day 10 of postnatal life. Owing to the difficulties in determining low ODC activities precisely, it is not exactly known when ODC activity attains adult values. On day 25 we measured 0.4-+ 0.3 nmol |4CO2formed/g brain/hr, at a time when SAM-DC activity had increased to 41 -+4 nmol 14CO2 formed/g brain/hr. 26 NI-Acetylspermidine accumulation rate was at this time (at about 1 nmol/g brain/hr) significantly higher than the in vitro determined ODC activity, suggesting that putrescine deriving from decarboxylation of ornithine was insufficient to maintain the observed spermidine turnover rate. Therefore re-utilization of the putrescine which is formed along the interconversion reaction is a requirement. According to the data in Table 3 this portion amounts to 60--80% of the total putrescine formed in the brains of animals which are 20 days or older. This is precisely the amount needed in addition to the ornithine derived putrescine, in order to maintain the observed rate of spermidine turnover. Around day 20 of postnatal life the observed putrescine pool is still considerably higher than in fully mature animals (Table 2), owing to the fully developed putrescine re-utilization system (Table 3), and an ODC activity assumedly still above adult levels. Together with an excessive SAM-DC activity maximum rates of spermidine formation are thus achieved during a short developmental stage. As was mentioned before, it is evident from the comparison of the activity of the various sources of the polyamine precursors that SAM-DC, not ODC, is the rate limiting enzyme of spermidine formation in the immature brain. It is evident now that in the mature brain, the total amount of putrescine formed is in control of spermidine synthesis, i.e. ODC activity and the interconversion reaction regulate in a concerted manner spermidine formation and degradation. Since there is no doubt that the rate of polyamine interconversion is limited by the rate of N L acetylation, and thus by the activity of the cytosolic acetyltransferase,9-u~ it is of great importance to establish those factors which are controlling the activity of this enzyme in brain under physiological conditions. The potential significance of the change from a SAM-DC controlled, to a putrescine controlled polyamine regulatory mechanism during brain maturation has previously been discussed. 2"18 It may therefore be sufficient to mention here without explanation that the regulatory system in physiologically mature brain seems (in contrast with the regulatory system of embryonal cells) suited to produce rapid changes in the rate of spermidine and spermine synthesis, in response to small changes of ODC activity. The observed excessive SAM-DC activity 12 and very high activities of spermidine and spermine synthase in vertebrate brains 12"13are prerequisites for the proposed regulatory system, and provide indirect support for our hypothesis. It can be assumed that the active regulation of polyamines by both synthetic and catabolic reactions is not restricted to brain cells, but may be found in other differentiated cell types as well. The new PAO inhibitors are the tool of choice for the detection of this type of regulatory mechanism.

REFERENCES I. Anderson T. R. and Schanberg S. M. (1972) Ornithine decarboxylase activity in developing rat brain. J. Neurochem. 19, 1471-1481. 2. Antrup H. and Seller N. (1980) On the turnover of polyamines spermidine and spermine in mouse brain and other organs. Neurochem. Res. 5, 123-143. 3. Bey P., Bolkenius F. N.. Seller N. and Casara P. (1985) N-2,3-Butadienyl-l,4-butanediamine derivatives: potent irreversible inactivators of mammalian polyamine oxidase. J. Med. Chem. 28, i-2. 4. Bolkenius F. N. and Seiler N. (1981) Acetylderivatives as intermediates in polyamine catabolism, int. J. Biochem. 13, 287-292. 5. Bolkenius F. N., Bey P. and Seiler N. (1985) Specific inhibition of polyamine oxidase in vivo is a method for the elucidation of its physiological role, Biochem. biophys. Acta 838, 69-76. 6. Hartree E. F. (1972) Determination of protein: a modification of the Lowry method that gives a linear photometric response. Analyt. Biochem. 48, 422-427. 7. Johanson C. E. (1980) Permeability and vascularity of the developing brain: cerebellum vs cerebral cortex. Brain Res. 190, 3-16.

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8. Mamont P. S., Bolkenius F., Seller N., Bey P. and Kolb M. (1984) Biochemical consequences of polyamine oxidase and N8-acetylspermidine deacetylase inhibition in cultured rat hepatoma (HTC) cells. Abstr. No. 6, Intern. Conference on Polyamines, Budapest, Hungary. 9. Pegg A. E., Seely J. E., P6s6 H., Della Ragione F. and Zagon I. S. (1982) Polyamine biosynthesis and interconversion in rodent tissues. Fed. Proc. 41, 3065-3072. 10. Persson L., Erwin B. G. and Pegg A. E. (1985) Spermidine/spermine NI-acetyltransferase: studies using a specific antiserum, in Recent Progress in Polyamine Research, pp. 287-296. Akademiai Kiado, Budapest. 11. Oeff K. and K6nig A. (1955) Das Blutvolumen einiger Rattenorgane und ihre Restblutmenge nach Entbluten bzw. Durchspiilung. Bestimmung mit P32-markierten Erythrocyten. Archs exp. Pharmac. 226, 98-102. 12. Raina A., Pajula R. L., EIoranta T. and Tuomi K. (1978) Synthesis of polyamines and S-adenosylmethionine in rat tissues and tumor cells: effect of DL-et-hydrazino-8-aminovaleric acid on cell proliferation. Adv. Polyamine Res. I, 75-82. 13. Raina A., EIoranta T., Hyv6nen T. and Pajula R. L. (1983) Mammalian propylamine transferases. Adv. Polyamine Res. 4, 245-253. 14. Schmidt G. L. and Cantoni G. L. (1973)Adenosylmethionine decarboxylase in developing rat brain. J. Neurochem. 20, 1373-1385. 15. Seller N. (1969) Enzymes. In Handbook ofNeurochemistry, Vol. 1, Ist edn (ed. Lajtha A.), pp. 325-468. Plenum Press, New York. 16. Seller N. (1970) Use of the dansyl reaction in biochemical analysis. Meth. biochern. Analysis 18, 259-337. 17. Seller N. (1981) Amide-bond forming reactions of polyamines. In Polyamines in Biology and Medicine (eds Morris D. R. and Marton L. J.), pp. 127-149. Marcel Dekker, New York, Basel. 18. Seller N. (1981) Turnover of polyamines. In Polyamines in Biology and Medicine (eds Morris D. R. and Marion L. J.), pp. 169-180. Marcel Dekker, New York, Basel. 19. Seller N. (1981) Polyamine metabolism and function in brain. Neurochern. Int. 3, 95-I 10. 20. Seller N. (1982) Handbook of Neurochemistrv. Vol. 1, 2nd edn (ed. Lajtha A.), pp. 223-255. Plenum Press, New York. 21. Seller N. and Bolkenius F. N. (1985) Polyamine reutilization and turnover in brain. Neurochem. Res. 4, 529-544. 22. Seller N. and Kn6dgen B. (1985) Determination of polyamines and related compounds by reversed phase HPLC. Improved separation systems. J. Chromatogr. 339, 45-57. 23. Seller N. and Lamberty U. (1975) Interrelations between polyamines and nucleic acids. Changes of polyamine and nucleic acid concentrations in the developing rat brain. J. Neurochem. 24, 5-13. 24. Seller N. and Lamberty U. (1975) Polyamines and nucleic acids in brains and hearts of newborn and adult autophagous and heterophagous vertebrates. Comp. Biochem. Physiol. 52B, 419--425. 25. Seller N. and Schmidt-Glenewinkel T. (1975) Regional distribution of putrescine, spermidine and spermine in relation to the distribution of RNA and DNA in the rat nervous system. J. Neurochem. 24, 791-795. 26. Seiler N., Bink G. and Grove J. (1980) Relationships between GABA and polyamines in developing rat brain. Neuropharmacology 19, 251-258. 27. Seller N., Bolkenius F. N., Bey P., Mamont P. S. and Danzin C. (1985) Biochemical significance of inhibition of polyamine oxidase. In Recent Progress in Polyamine Research, pp. 3115-319. Akademiai Kiado, Budapest. 28. Seller N., Bolkenius F. N. and Kn6dgen B. (1985) The influence of catabolic reactions on polyamine excretion. Biochem. J. 225, 219-226. 29. Seller N., Bolkenius F. N., Kn6dgen B. and Mamont P. S. (19811) Polyamine oxidase in rat tissues. Biochirn. biophys. Acta 615, 480-488. 311. Seller N., Bolkenius F. N. and Rennert O. M. (1981) Interconversion, catabolism and elimination of the polyamines. Med. Biol. 59, 334-346. 31. Seller N., Bolkenius F. N. and Sarhan S. (1981) Formation of acetylpolyamines in the liver of fasting animals. Int. J. Biochem. 13, 1205-1214. 32. Tabor H., Tabor C. W. and De Meis L. (1971) Chemical synthesis of N-acetyl-l,4-diaminobutane. Nt-acetylspermidine and N~-acetylspermidine. Meth. Enzymol. 17b, 829-833. 33. Winer B. J. (1971) Design and analysis of single-factor experiments. In Statistical Principles in Experimental Design. McGraw-Hill, New York.