Polyamine metabolism in reversible cerebral ischemia: effect of α-difluoromethylornithine

Brain Research, 453 (1988) 9-16

9

Elsevier BRE 13659

Polyamine metabolism in reversible cerebral ischemia: effect of a-difluoromethylornithine W u l f P a s c h e n I , G a b r i e l e R 6 h n 1 , C l a u s O . M e e s e 2,

Bogdan Djuricic 3 and Rainald Schmidt-Kastner 1 IMax-Planck-lnstitute for Neurological Research, Department of Experimental Neurology, Cologne (F. R. G.), 2Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart (F.R.G.) and 3Institute of Biochemistry, Faculty of Medicine, University of Belgrade, Belgrade (Yugoslavia) (Accepted 22 December 1987)

Key words: Cerebral ischemia; ct-Difluoromethylornithine; Ornithine decarboxylase; Polyamine metabolism; Putrescine; Rat

Severe forebrain ischemia was produced in rats by occluding both carotid and vertebral arteries. Following 30 min ischemia brains were recirculated for 8 or 24 h. Twelve animals subjected to 8 or 24 h recirculation (n = 6, each group) were given a-difluoromethylornithine (DFMO; injected intraperitoneally) immediately before recirculation. At the end of the experiments brains were frozen and samples were taken from the cerebellum, cortex, caudatoputamen and hippocampus. Samples from the left hemisphere were used for measuring ornithine decarboxylase (ODC) activity, and those from the right hemisphere for determining putrescine profiles. During recirculation ODC activity increased markedly in all brain structures, the most pronounced change being in the caudatoputamen after 8 h recirculation. Putrescine increased drastically after 8 h and even more after 24 h recirculation. DFMO-treatment significantly reduced ODC activity after 8 h recirculation and following 24 h recirculation. Putrescine, however, was significantly reduced following 24 h but not after 8 h recirculation. The discrepancy between reduction in ODC activity and putrescine revels in DFMO-treated animals was most prominent in the hippocampus after 8 h recirculation: here DFMO reduced ODC activity to control values without affecting putrescine levels. The results suggest that the observed overshoot in putrescine formation following ischemia is only partly caused by activation of ODC.

INTRODUCTION Polyamines (putrescine, spermidine and spermine) are known to play a f u n d a m e n t a l role in the regulation of cellular growth processes, including pre- or neonatal growth, cell proliferation and regeneration 8A2,15'26'28'29'34'37. Polyamine m e t a b o l i s m is regulated by changes in the activity of two key enzymes, namely ornithine decarboxylase ( O D C ; E C 4.1.1.17) which catalyses the d e c a r b o x y l a t i o n of ornithine to putrescine, and 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) which catalyses the decarboxylation of S - a d e n o s y l m e t h i o n i n e to decarboxylated S-adenosylmethionine. Recently in the brain an increase in the activity of O D C has been shown to be a c o m m o n response to

different pathological stimuli 1'9. The most pronounced changes in p o l y a m i n e metabolism were observed in reversible cerebral ischemia 1°'17. O D C activity is m a r k e d l y increased during recirculation following cerebral ischemia. Postischemic changes in S A M D C activity d e p e n d , however, on the animal species studied: in the rat, 30 min occlusion of both carotid and vertebral arteries produces a severe depression in S A M D C activity for several days ~°. In contrast, in the m o n k e y brain, the activity of S A M D C is increased considerably after 1 h complete cerebral ischemia and p r o l o n g e d recirculation (from about 12 h onwardsl7). Polyamine profiles have been m e a s u r e d recently during and following cerebral ischemia 24'25. The most prominent change was a m a r k e d increase in putres-

Correspondence: W. Paschen, Max-Planck-Institute for Neurological Research, Department of Experimental Neurology, Ostmerheimer Strasse 200, 5000 KOln 91, F.R.G. 0006-8993/88/$03.50 (~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)

1[I cine during recirculation. This increase in putrescine has been suggested to be caused by the postischemic activation of ODC and inhibition of SAMDC 24. The present series of experiments was designed to study the effect of a-difluoromethylornithine (DFMO: an irreversible inhibitor of ODC z:) on the ischemia-induced changes in polyamine metabolism. The results suggest that the postischemic overshoot in putrescine formation is only partly caused by the increase in ODC activity. MATERIALSAND METHODS

Anirnal preparation Thirty-six rats of both sexes and weighing 270-350 g were used. Reversible cerebral ischemia was induced in 30 animals using the 4-vessel occlusion model described by Pulsinelli and Brierley 33 with modifications (Schmidt-Kastner et al., in preparation). On Day 1, animals were anesthetized (1.5% halothane, 70% nitrous oxide, 30% oxygen), both vertebral arteries were occluded by electrocoagulation, and rats were starved overnight. On Day 2, animals were reanesthetized (1.5% halothane, 70% nitrous oxide, 30% oxygen). An artery catheter was inserted into the tail for the sampling of blood (for measuring of pO> pCO 2, pH, hematocrit and glucose) and for the recording of blood pressure. The body temperature was controlled and maintained at 37 °C. The electroencephalogram (EEG) was recorded from the scalp. Both common carotid arteries were exposed. Halothane was then reduced to 0.5% until the physiological parameters stabilized spontaneously. Animals in which the physiological parameters did not stabilize spontaneously were discarded from the present study. After control o f physiological parameters anesthesia was discontinued and both common carotid arteries occluded for 30 min using aneurysm clips. Only those animals were selected for the present study in which: (a) spontaneous ventilation was sustained during ischemia; and (b) the E E G flattened during the first 60 s of ischemia and remained isoelectric throughout. Following 30 rain cerebral ischemia brains were spontaneously recirculated by opening the aneurysm clips. At the end of the experiments (after 30 rain ischemia or following 8 or 24 h recirculation) animals were reanesthetized (1.59/- halo-

thane, 70% nitrous oxide, 30% oxygen), physiological parameters were controlled and corrected it necessary, and brains were frozen in situ with liquid nitrogen 32. Sham-operated animals (n = 6) in which both vertebral arteries but not the carotid arteries were occluded served as controls.

D FM O-treatment In 12 animals (subjected to 31,)min ischemia and 8 or 24 h recirculation, n = 6 each group) DFMO was injected intraperitoneally to inhibit the postischemic activation of polyamine metabolism. DFMO was dissolved in water (175 mg/ml) and the pH adjusted to 7.4 before use. In all animals DFMO (500 mg/kg) was injected immediately at the end of the ischemic period. Treated animals to be subjected to 24 h recirculation received a second dose of DFMO (500 mg/kg) after 12 h recirculation. DFMO did not produce any acute effect on the hematocrit, blood glucose levels or blood gases.

Analysis of putrescine and ODC Brains were removed from the skull in a low temperature cabinet at -2() °C. Tissue samples of about 10-15 mg each were taken from the cerebellum, cerebral cortex, caudatoputamen and hippocampus of both hemispheres. Samples taken from the right hemisphere were used for measuring putrescine levels t~z~ and those from the left hemisphere for measuring the activity of ODC. ODC activity was assessed quantitatively in tissue samples by measuring the release of t4CO~ from L-[1-14C]ornithine (NEN Chemicals, Dreieich, F.R.G.: spec. act. 52.6 mCi/ mmol). Brain samples were homogenized at 4 °C with 10 vols. of Tris-buffer (50 mM, pH 7.2, supplemented with 2 mM dithiothreitol (DTT)). The assay was carried out in sealed tubes containing center wells. The test mix was composed of: brain homogenate (4.5 mg of tissue) in Tris/DTT-buffer, supplemented with pyridoxalphosphate (50 IzM), in a total volume of 140 t¢1. Samples were preincubated in the presence or absence of DFMO (1.8 raM) at 37 °C for 15 min for measuring both the 'non-specific' and 'specific' (ODC-induced) decarboxylation of ornithine. After preincubation the reaction was started by the addition of [HC]ornithine (68,uM, 52.6 mCi/mmol). After 60 rain the reaction was terminated by injecting 100 !~1 perchloric acid (0.6 N) into the test rnix.

11 14CO2 released by ODC during incubation was trapped on a filter paper in the center well (impregnated with 150/~1 Hyamine, 1 M in methanol). After further incubation at 37 °C for 30 min tubes were opened, the filter paper put in a scintillation vial containing 10 ml Econofluor (NEN Chemicals, Dreieich, F.R.G.) supplemented with 1 ml methanol, and the radioactivity measured in a liquid scintillation counter. ODC activity in the tissue samples was calculated by subtracting the 'non-specific' reaction (14CO2 release in the presence of DFMO) from the total reaction (IaCO= release in the absence of DFMO). Under these conditions release of k4CO~ from [14C]ornithine was linear with time.

pound was assessed by studying the chromatographic and spectroscopic properties. Both thin layer chromatography (silica gel plates; mobile phase = n-butanol/acetic acid/water (3:1:1)) and 13C-NMR spectroscopy revealed high purity of DFMO.

Preparation of DFMO

In the present study release of 14CO2 from [14C]ornithine was measured both in the presence and absence of DFMO. In control animals the 'non-specific' activity (measured in the presence of DFMO) amounted to 0.46 + 0.03, 0.45 + 0.03, 0.41 + 0.06 and 0.44 + 0.11 nmol/g/h in the cerebellum, cerebral cortex, caudatoputamen and hippocampus, respectively. This 'non-specific' activity did not change sig-

Statistics Statistical comparison of ODC activity and content of putrescine between all groups studied was performed using analysis of variance (ANOVA). Differences between groups were calculated using the Scheffe's F-test. Values given are means + S.E.M. RESULTS

Racemic (_+)-a-(difluoromethyl)ornithine hydrochloride monohydrate was prepared in 6 steps from L-ornithine hydrochloride as described previously 3"4. The salt obtained was purified by 3 recrystallizations from water/methanol. The melting point of the pure product was 247 °C (references Bey and Vevert 3 and Bey et al.4: m. p. 183 °C). The purity of the final corn-

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Fig. 1. Effect of DFMO-treatment on the concentration of putrescine (left) and activity of O D C (right) in the cerebral cortex of control rats and animals subjected to 8 h or 24 h recirculation following 30 min forebrain ischemia. D F M O (500 mg/kg, injected intraperitoneally) was given at the end of ischemia and, in the case of 24 h recirculation, a second dose after 12 h recirculation. Tissue samples of about 10-15 mg each were taken from the cerebral cortex of both hemispheres. Samples taken from the right hemisphere were used for measuring putrescine levels and those from the left hemisphere for measuring the activity of ODC. Values are means + S.E.M. Statistically significant differences between experimental groups and controls are indicated by: b.c compared to control animals (P ~< 0.01 or P ~< 0.001, respectively); d.f compared to untreated animals (P ~< 0.05 or P ~< 0.001, respectively).

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although the relative increase in O D C activity was much higher in the caudatoputamen than in the hippocampus (20.9-fold and 4.2-fold. respectively). Following 24 h recirculation O D C activity decreased substantially in the cerebellum, only slightly in the cortex and caudatoputamen, but increased further in the hippocampus. Putrescine content, in contrast, increased in all brain structures studied during prolonged recirculation: the highest levels were measured in the caudatoputamen and hippocampus, less so in the cortex and even less in the cerebellum. The effect of DFMO-treatment on regional postischemic O D C activity and putrescine levels depended on the time period of recirculation and varied considerably in different brain structures (Figs. 1-4). Following 8 h recirculation D F M O produced only a slight decrease of putrescine content but drastically reduced O D C activity. This discrepancy between O D C and putrescine in DFMO-treated animals was most pronounced in the hippocampus; here D F M O did not have any effect on putrescine content although O D C activity was reduced to control levels (Fig. 4). Following 24 h recirculation O D C activity and putrescine levels were severely reduced in D F M O -

nificantly during or following ischemia in untreated or treated animals, which is in accordance with the results of Dienel et al. 10. The regional activities of O D C and levels of putrescine in control brains and brains recirculated for 8 or 24 h in the presence or absence of D F M O are summarized in Figs. 1-4. In the brains of control animals no significant regional differences in putrescine content or O D C activity were observed with the exception of the hippocampus in which O D C activity was significant higher than that measured in the caudatoputamen (P ~< 0.01). Thirty minute forebrain ischemia did not produce any significant changes in regional putrescine levels or O D C activity (data not shown). In untreated animals the activity of O D C and content of putrescine increased markedly during recirculation in all brain structures studied (Figs. 1-4). Following 8 h recirculation the most pronounced increase in O D C and putrescine was apparent in the caudatoputamen. The regional increase in putrescine levels, however, did not correlate closely with the increase in O D C activity; i.e. the relative increase in putrescine was comparable in the hippocampus and caudatoputamen (5.1-fold and 7.2-fold, respectively)

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Fig. 2. Effect of DFMO-treatment on the concentration of putrescine (left) and activity of ODC (right) in the caudatoputamen of control rats and animals subjected to 8 h for 24 h rccirculation following 30 min forebrain ischemia. For details see legend to Fig. 1. Values are means + S.E.M. Statistically significant differences between experimental groups and controls are indicated by: b.c compared to control animals (P ~<0.01 or P ~<0.001, respectively); d'fcompared to untreated animals (P ~<0.05 or P ~ 0.001, respectively).

13 treated animals: O D C activity was reduced to or even below (hippocampus) control values. In the cerebellum, cerebral cortex and hippocampus of DFMO-treated animals putrescine levels were markedly reduced (from 8 h to 24 h of recirculation) and were only slightly above but not statistically significant different from those found in sham-operated control animals. In the caudatoputamen, in contrast, putrescine increased further in treated animals from 8 to 24 h recirculation.

days recirculation. Following 8 h recirculation the relative increase in O D C activity has been shown to be most pronounced in the caudatoputamen, less so in the cerebral cortex and even less in the hippocampus. The results of the present study are similar to those described by Dienel et al. ~0. There are, however, two fundamental differences: in the present study O D C activity was still high in the caudatoputamen after 24 h recirculation (about 1500% of the control value), and in the hippocampus the O D C activity did not drop following prolonged recirculation but even increased further (from 415% of control values to 810% with 8 h and 24 h recirculation periods, respectively). These differences in the regional time course of O D C activity during recirculation may be caused by differences in the experimental procedure or, more likely, by differences in the O D C assay. O D C activity in tissue samples is usually measured in the post-mitochondrial supernatants to remove 'non-specific' activity (ODC-independent formation of 14CO2 from [1JaC]ornithine) located in the mitochondria. Secondly, a high concentration of ornithine is used to guarantee substrate saturation of

DISCUSSION It has been shown that reversible cerebral ischemia induces a marked increase in ornithine decarboxylase ( O D C ) activity 9,1°:7. Regional changes in O D C activity have been measured recently in rat brains during and following cerebral ischemia produced in the same way as in the present study namely, with the 4-vessel occlusion model 33. O D C activity is unchanged during ischemia, considerably reduced during the first 2 h of recirculation, and markedly increased during prolonged recirculation l°, peaking at about 8 h recirculation and a second time at about 3

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Fig. 3. Effect of DFMO-treatment on the concentration of putrescine (left) and activity of ODC (right) in the hippocampus of control rats and animals subjected to 8 or 24 h recirculation following 30 min forebrain ischemia. For details see legend to Fig. 1. Values are means +_S.E.M. Statistically significant differences between experimental groups and controls are indicated by: ~.b.ccompared to animals (P ~<0.05, P ~<0.01 or P ~<0.001, respectively); e.ecompared to untreated animals (P ~<0.01 or P ~<0.00l, respectively).

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ODC and to minimize the effects of any changes in the endogenous concentration of ornithine or putrescine (which is a weak competitive inhibitor of ODC 27) during postischemic recirculation. In the present series of experiments whole tissue homogenates were used for measuring ODC activity and the assay was carried out in the presence of 68 ~M ornithine, which is in the range of the K mfor ornithine 7.23. 'Non-specific' formation of 14CO2 from [1J4C]ornithine did not interfere in our test system since all samples were measured in duplicate in the presence and absence of DFMO, thus permitting one to calculate the 'specific' activity. In addition, use of whole tissue homogenates is considered to be advantagous as long as the exact localization of the increased ODC activity (observed during recirculation after ischemia) is not known. It has been shown recently that in the brain ODC activity is localized mainly in the particulate and much less in the soluble fraction5. It cannot be excluded, therefore, that only a fraction of the total ODC activity is measured when post-mitochondrial supernatants are used for the assay. The advantage of using low ornithine concentrations (as in the present paper) is obvious; the specific

activity of ornithine, and therefore the sensitivity of the assay, can be increased considerably, thus permitting small tissue samples to be analyzed; in addition, a change in the K m of ODC for ornithine cannot be detected under substrate-saturation conditions. There are, however, negative aspects which shall be discussed in detail, namely: (a) a possible influence of endogenous ornithine or putrescine on the ODC assay; and (b) a possible change of [14C]ornithine concentration in the test tube during measurement of samples exhibiting high ODC activity. (a) Any significant influence of endogenous ornithine or putrescine on the ODC assay can be ruled out here, since the concentration of both substrates was measured in tissue samples taken from the same brains as those in which ODC activity was analyzed. Ornithine levels were evaluated semiquantitatively in the same HPLC-chromatograms as putrescine. In all brain samples studied, endogenous ornithine was below 100/~M, i.e. below about 3pM in the ODC assay. In addition, the highest putrescine levels found following prolonged recirculation were about 200 nM. Thus, in the ODC assay putrescine never exceeded about 6 ~M, which is far below the Ki of putrescine for O D e 27. (b) The highest [l-~4C]ornithine decar-

15 boxylating activity found in any of the brain samples studied was 9.8 pmol/mg/h (hippocampus, 24 h recirculation: 9.4 pmol/mg/h 'specific' activity and 0.4 pmol/mg/h 'unspecific' activity (in the presefice of DFMO)), which is equivalent to 44.1 pmol/4.5 mg/h (4.5 mg of tissue used in the assay). Since each test tube contained 9.5 nmol [14C]ornithine, only less than 0.5% of the substrate was decarboxylated during incubation of samples exhibiting high O D C activity. The most striking aspect of the present study was the dissociation between the effect of DFMO-treatment on the O D C activity and the concentration of putrescine during recirculation. This discrepancy was most prominent in the hippocampus following 8 h recirculation; in DFMO-treated animals putrescine levels were identical to those measured in untreated animals (54.54 _+ 6.77 and 51.21 _+ 5.28 nmol/g, respectively) although ODC activity was markedly reduced (from 3.53 + 0.62 to 0.95 _ 0.29 nmol/g/h). Further, following 24 h recirculation DFMO-treatment reduced the ODC activity in the hippocampus to below control values (0.43 _+ 0.04 compared to 0.85 _+ 0.14 nmol/g/h in controls) and the putrescine levels nearly to control levels, whereas putrescine concentration was high in the caudatoputamen. The observed dissociation between O D C activity and putrescine levels in DFMO-treated animals strongly suggests that activation of O D C during recirculation is not the only cause for the postischemic increase in putrescine. Besides activation of ODC, inhibition of S-adenosylmethionine decarboxylase (SAMDC) may be responsible for the postischemic overshoot in putrescine formation. SAMDC activity is severely depressed in the rat brain during recirculation following ischemia 1°. Since SAMDC is necessary for the synthesis of spermidine from putrescine and spermine from spermidine, a low SAMDC activity is considered to influence putrescine levels 13. It cannot be ruled out however, that other enzymes of polyamine metabolism are involved in the postischemic increase in putrescine; i.e. the enzymes spermidine/ spermine Nl-acetyltransferase: it has been shown that carbon tetrachloride poisoning of the mouse

REFERENCES 1 Ali, S.F., Newport, G.D., Slikker, W. and Bondy, S.C.

liver produces a marked increase in the activity of spermidine/spermine Nl-acetyltransferase 3°. It has been suggested recently that the pathological disturbances in polyamine metabolism observed following cerebral ischemia, especially the postischemic increase in putrescine, contribute to the manifestation of ischemic cell injury25; this is most evident in the CAt-subfield of the hippocampus. Here the postischemic putrescine level correlates closely with the density of ischemic neuronal damage observed following prolonged recirculation, and the postischemic increase in putrescine is already apparent after 8 h recirculation, i.e. at a time when the cells are still morphologically and functionally intact and exhibit a physiological energy-state 2'16'35. Putrescine has been shown recently to play a fundamental role in the control of Ca2+-related events at the cell membrane; it has been suggested that putrescine activates the flux of calcium into the cell 18'19, that it triggers the release of neurotransmitters from synaptosomes following stimulation 6'14m, and that it contributes to the disturbances in the blood-brain barrier following cold injury 2°'36. It is, therefore, conceivable that putrescine is involved in the pathological sequelae of events finally leading to ischemic damage to neurons. This hypothesis can clearly be answered by blocking completely the postischemic increase in putrescine. The results of the present series of experiments indicate that DFMO-treatment produces only a slight effect on the putrescine levels during early recirculation. It will be interesting to study whether high doses of DFMO given at short time intervals during recirculation or even before induction of ischemia have any effect on the ischemic damage to neurons. ACKNOWLEDGEMENTS The excellent technical assistance of Mrs. S. Beck, Mrs. F. Haar, Mrs. C. Magendanz and Mrs. J~. Zohren is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft, Grant Pa 266/2-4. The travel expenses of B.D. were covered by a grant from European Science Foundation. Effect of trimethyltin on ornithine decarboxylase in variou,, regions of the mouse brain, Toxicol. Lett., 36 (1987) 67-72. 2 Arai, H., Passonneau, J.V. and Lust, W.D.. Energy me-

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