Polyamine reutilization and turnover in brain

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<ul><li><p>Neurochemical Research, Vol. 10, No. 4, 1985 </p><p>POLYAMINE REUTILIZATION AND TURNOVER IN BRAIN </p><p>NIKOLAUS SEILER AND FRANK N. BOLKENIUS Merrell-Dow Research Institute Strasbourg Center </p><p>16 rue d'Ankara 67084 Strasbourg France </p><p>Accepted November 19, 1984 </p><p>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. </p><p>INTRODUCTION </p><p>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. </p><p>529 0364-3190/85/0400-0529504.50/0 9 1985 Plenum Publishing Corporation </p></li><li><p>530 SEILER AND BOLKENIUS </p><p>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 &gt;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. </p><p>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. </p><p>EXPERIMENTAL PROCEDURE </p><p>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. </p><p>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. </p><p>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. </p><p>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). </p><p>A portion of this homogenate was mixed with an equal volume of 0.4 M HC104 and the supernatant was used for polyamine determinations. </p><p>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). </p><p>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- </p></li><li><p>POLYAMINE REUTILIZATION AND TURNOVER 531 </p><p>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). </p><p>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). </p><p>RESULTS </p><p>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. </p><p>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. </p><p>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. </p><p>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. </p></li><li><p>532 SEILER AND BOLKENIUS </p><p>9 NS- ( 2-Carboxyethyl ) -sPe rmldlne SPERMINE I SDermlc acid </p><p>Decarboxyl. SAM 2 s~ \ N I-Acetylsp ermine </p><p>9 ................... N8-Acetyl spe rmidlne 9 </p><p>N_ACety lput rean lne </p><p>SAM </p><p>Put rean lne : SPERMIDINE ~ Isoput~eanlne 1scram </p><p>y, CO2 [ :3 N I -Acetylspermidlne </p><p>co~ jae~PUTRESCiN/ ~ ...... Yl~asoP ~ ...... </p><p>ORNITHINE / 6 \ </p><p>G1 utemic semialde hyde 02&amp;~ NI-Acet ylputrescine / </p><p>4 ~[~l Succinic 2-Oxoglutarate ~ semial dehyde </p><p>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) </p><p>10 = Ornithine:2-oxoacid aminotransferase 11 = 4-aminobutyrate: 2-oxoacid aminotransferase (GABA-T) 12 = Glutamate decarboxylase (GAD). </p><p>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- </p></li><li><p>I </p><p>I i i i </p><p>! </p><p>! </p><p>o </p><p>POLYAMINE REUTIL IZAT ION AND TURNOVER </p><p>! i I i i I i I i I i I i I I </p><p>t--- </p><p>r </p><p>I i I </p><p>~ ~ ~ ,r </p><p>U!D~] B/lOW u </p><p>I l l </p><p>z </p><p>~l =o </p><p>9 I I </p><p>' N OI..LV~IJ.N33NO3 </p><p>J </p><p>o </p><p>m </p><p>= 00 </p><p>m </p><p>- O </p><p>533 </p><p>L </p><p>( </p><p>( ( </p><p>I L </p><p>ii </p><p>1 </p><p>I. 0 ' </p><p>| I ~ o </p><p>u!o~8 B / IOWU </p><p>O0 </p><p>, I ! </p><p>= = :i o o = ',', </p><p>, , </p><p>i i i | I I I I i I </p><p>I </p><p>|1 </p><p>\ \ ,, </p><p>-,,0 x,~ </p><p>I I ! 0 </p><p>'NOI177 </p><p>J o </p><p>0 </p><p>.,d O </p><p>~~ </p><p>O~ ~ . ~ , .d z ~ ~ </p><p>m </p><p>_ ~ ~:~ </p><p>It: ~ 'o~ </p><p>o~ </p><p>~ ~.~ </p></li><li><p>534 SEILER AND BOLKENIUS </p><p>rz~ </p><p>-6 E c </p><p>v </p><p>Z 0 l,- </p><p>n. l- z w </p><p>z o o </p><p>t.l.J Z </p><p>rr ILl rl O3 &gt;- I--- W &lt; -Z </p><p>30 </p><p>20 </p><p>10 </p><p>0 </p><p>HEMISPHERES </p><p>.0" d j </p><p>I I </p><p>30 </p><p>20 </p><p>I0 </p><p>0 </p><p>CEREBELLUM </p><p>| I 0 24 </p><p>TIME AFTER i.p. INJECTION </p><p>BRAINSTEM </p><p>...s-"Yg"* I ! </p><p>MEDULLA </p><p>/ . . ; , .o- </p><p>I J </p><p>0 24 </p><p>OF MDL72527 (h ) </p><p>0 CONTROL 9 DFMO-TREATED [/X]=nrnol /g broin / h </p><p>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. </p><p>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. </p><p>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. </p></li><li><p>POLYAMINE REUTILIZATION AND TURNOVER 535 </p><p>D o E c </p><p>I0 I HEMISPHERES 1-~ SALINE </p><p>5 I ~ MDL 72527 I DFM0 </p><p>MDL 72 527 0 + DFMO </p><p>IO </p><p>5 </p><p>O </p><p>15 - </p><p>I0 </p><p>~ EM CEREBELLUM </p><p>_L </p><p>5 5 </p><p>15- </p><p>! </p><p>MEDULLA </p><p>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. </p><p>The animals were decapitated 24 hr after MDL 72527 administration. The DFMO-treated groups had access to this drug until sacrifice. </p><p>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)). </p><p>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 </p></li><li><p>536 SE ILER AND BOLKENIUS </p><p>H E MISPI.;,IERE S </p><p>0.5 </p><p>0 </p><p>T BRAINSTEM </p><p>2 - . t </p><p>1.5 </p><p>I </p><p>0.5 </p><p>o __ :i:i:~:~: </p><p>EREBELLUM .-T.-- </p><p>I </p><p>0.5 </p><p>SALINE </p><p>MDL 72527 </p><p>I {~FMO MDL 72527 + DFMO </p><p>4. - L MEDULLA </p><p>I 3.5 T * 3 </p><p>2.5 </p><p>2 </p><p>1.5 </p><p>I </p><p>0.5 - </p><p>o o - </p><p>F ie . 5. Spermidine/Spermine ratios in four brain parts of mice. Treatment schedule and analytical procedures were identical with those described in the </p><p>legend to Fig. 4. An asterisk indicates a statistically significant difference (P -&lt; 0.05) between treated and control group. Student's t-test (two tailed)). </p><p>putrescine levels was m agreement with previously reported findings (18). Spermidine and spermine levels were not affected by treatment with the GABA-T inhibitors. </p><p>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 </p></li><li><p>POLYAMINE REUTILIZATION AND TURNOVER </p><p>PUTRESCINE CONCENTRATION </p><p>537 </p><p>c (D </p><p>rn </p><p>-5 E c </p><p>c </p><p>E rn </p><p>-5 E c </p><p>200 </p><p>150 </p><p>I00 </p><p>50 </p><p>0 </p><p>30 </p><p>20 </p><p>I0 </p><p># NI-ACETYLSPERMIDINE </p><p>/ </p><p>I I i I I </p><p>0 2 4 6 8 </p><p>l I i i l </p><p>CONCENTRATION </p><p>/ I / </p><p>I I ' i J </p><p>0 2 4 6 8 </p><p>TIME AFTER i.p. INJECTION OF MDL 72527 (h) </p><p>FIG. 6~ The effect of treatment with GABA-T inhibitors on putrescine concentrations and the rate of Nl-acetylspermidine accumulation in mouse brain. </p><p>Left panel (filled symbols) treatment with 3,-vinylGABA. Right panel (empty symbols) treatment with wacetylenicGABA. </p><p>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). </p><p>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. </p></li><li><p>538 SEILE...</p></li></ul>

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