EXPERIMENTAL
NEUROLOGY
117,210-215
(1992)
Activity of Ornithine Decarboxylase and S-Adenosylmethionine Decarboxylase in Transient Cerebral Ischemia: Relationship to the Duration of Vascular Occlusion GABRIELERGHN,MICHAELSCHLENKER,ANDWULFPASCHEN Max-Planck
Institute
for Neurological
Research,
Department
Copyright All rights
Inc. reserved.
cologne,
Germany
underlyand pro-
INTRODUCTION The synthesis of the polyamines spermidine and spermine and their precursor putrescine is controlled by the activity of the two key enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). Transient cerebral ischemia induces marked changes in polyamine synthesis, namely a sharp increase in ODC and decrease in SAMDC activity (3,6,10, 15). The result is an overshoot in the formation of putrescine, the product of the ODC reaction, because synthesis of putrescine is activated whereas the interconversion of putrescine into spermidine and spermine is suppressed due to low SAMDC activity. Since postischemit putrescine levels correlate closely with the density of neuronal necrosis (15) and putrescine mediates neurotransmitter release from nerve endings and calcium fluxes at the cell membrane (2, 8, 12) it has been suggested that this compound is involved in the manifestation of ischemia- or hypoglycemia-induced neuronal necrosis (14,18). It has been shown previously that during recirculation putrescine levels correlate closely with the duration of ischemia (16), but no information has been available up to now to indicate whether any such relationship exists between the duration of ischemia and the postischemic ODC and SAMDC activity. In the present series of experiments, therefore, postischemic changes in ODC and SAMDC activity were studied in relation to the duration of ischemia. Animals were subjected to 2,4,6,8, or 10 min of ischemia and 8 or 24 h of recovery. A threshold ischemic duration for ODC induction was observed, the duration of ischemia needed to produce a significant increase in ODC activity being 4 min in the hippocampus, 6 min in the cortex and striaturn, and 8 min in the thalamus. 210
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Neurology,
information about the molecular mechanisms ing stress-induced changes in gene expression tein synthesis. o 1992 Academic PWSS, h.
Mongolian gerbils were anesthetized with halothane and forebrain ischemia was induced by occluding both common carotid arteries. After 2,4,6,8, or 10 min of vascular occlusion clips were removed and animals allowed to recover for 8 or 24 h. At the end of the experiments animals were reanesthetized and their brains frozen in situ. Tissue samples were taken from the cerebral cortex, striatum, hippocampus, and thalamus for determination of ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (SAMDC) activity by measurement of the release of 14C0, from [14C]ornithine and 5’-[‘4C]adenosylmethionine, respectively. A transient increase in ODC activity was found after 8 h of recirculation following cerebral ischemia in all brain structures studied. ODC activity was significantly increased after 8 h of recirculation in the hippocampus of animals subjected to 4 min of ischemia, in the cortex and striatum after 6 min of ischemia, and in the thalamus after 8 min of vascular occlusion. ODC activity had already reached a plateau in the hippocampus after 4 min of vascular occlusion and in the cortex, striatum, and thalamus after 8 min, since there is no further increase in activity even after 10 min of ischemia. After cerebral ischemia and 24 h of recirculation ODC activity returned to control levels throughout the forebrain regardless of the duration of ischemia. SAMDC activity was significantly reduced after 8 h of recirculation following 4 to 10 min of ischemia in the cortex and 8 min of ischemia in the striatum. After 24 h of recirculation a significant reduction of SAMDC activity was measured in the cortex of animals subjected to 8 or 10 min of ischemia, in the striatum following 6 or 10 min of ischemia, and after 10 min of ischemia in the hippocampus. In control animals the SAMDUODC activity ratio (as an index of the extent of the disturbance of polyamine synthesis) ranged between 54.3 f 17.2 (thalamus) and 172.4 + 54.7 (cortex). After only 4 min of ischemia and 8 h of recirculation this ratio declined to 2.8 + 1.7 in the vulnerable hippocampus but was still between 26.2 + 18.4 (cortex) and 39.9 + 50.5 (striaturn). It is suggested that studying ischemia-induced changes in ODC and SAMDC activity may provide new 0014-4666192
of Experimental
ODC
MATERIALS
AND
AND
SAMDC
IN
TRANSIENT
CEREBRAL
211
ISCHEMIA
METHODS
8h recirculation
Animal Experiments
Striatum
Sixty-six male Mongolian gerbils (Meriones unguiculatus) weighing 50-80 g were used. Both common carotid arteries were occluded under anesthesia (1.5% halothane in 70% N,O and 30% 0,) with aneurysm clips for 2,4,6,8, or 10 min. During anesthesia rectal temperature was measured and maintained at 375°C by a feedback-controlled infrared heating system. Sham-operated animals in which both carotid arteries were exposed but not occluded served as controls. At the end of ischemia the clips were removed to allow reperfusion and the skin incision was sutured. After 8 or 24 h of recirculation the animals were reanesthetized and the brains frozen in situ with liquid nitrogen. Brains were removed from the skull at -20°C. Tissue samples were taken from the cerebral cortex, the striatum, the hippocampus, and the thalamus for measurement of enzyme activities.
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All data are expressed as means f SD. Statistical analysis was performed using Student’s t test with the Bonferroni corrections for multiple comparisons. A P value of less than 0.05 was considered to indicate significant differences. RESULTS
The activities of ODC measured after 2 to 10 min of ischemia and 8 or 24 h of recirculation are shown in Fig. 1. In the brains of control animals no regional differences in ODC activity could be detected: average enzyme activity amounted to 0.43 f 0.11, 0.36 + 0.11, 0.40
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Enzyme Analysis Determination of the activity of ornithine decarboxylase (ODC, E.C. 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMDC, E.C. 4.1.1.50) was performed as described elsewhere (19, 21) with some modifications (15). In brief, brain samples were homogenized in 25 vols (w/v) Tris/HCl buffer (50 mM, pH 7.2, supplemented with 5 mM DTT and 0.1 mM EGTA). The test mix for the measurement of ODC activity was composed of brain homogenate (4 mg of tissue), pyridoxal-5-phosphate (54 PLM), and L-[1-14C]ornithine (74 pLM, 51.6 mCi/mmol, New England Nuclear Corporation) in a total volume of 130 ~1. For the determination of SAMDC activity brain homogenate (1 mg of tissue), putrescine (2.5 mM), EDTA (1 n&Q and S-[carboxy-14C]adenosylL-methionine (0.2 mM, 16.7 mCi/mmol, New England Nuclear Corporation) were mixed yielding a final volume of 140 ~1. Enzyme activities were quantified by measuring the release of 14C0, from [14C]ornithine and S-[14C]adenosylmethionine for ODC and SAMDC, respectively.
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FIG. 1. ODC activity after 2-10 min of ischemia and 8 h (upper part) or 24 h (lower part) of recirculation. Data are expressed as nanomoles per gram tissue per hour (means t SD). **P < 0.01, ***P < 0.001, control, cf. experimental animals.
i 0.18,0.52 +- 0.11 nmol/g/h in the cortex, striatum hippocampus, and thalamus, respectively. After 8 h of recirculation there was a significant increase in enzyme activity; the temporal profile varied however in different brain structures. Postischemic ODC activity was significantly increased in the hippocampus after only 4 min of ischemia (to 9.01 f 5.36 nmol/g/h, P < 0.01, cf. controls), in the cortex and striatum after 6 min of vascular occlusion (to 3.16 + 1.83 and 2.00 -t 1.12 nmol/g/h, respectively, P < 0.01, cf. controls), and in the thalamus after 8 min of ischemia (to 4.07 + 0.63 nmol/g/h, P -C 0.001, cf. controls). When the duration of ischemia was extended ODC activity was shown to have reached a plateau, in the hippocampus already after 4 min of ischemia and in the cerebral cortex, striatum, and thalamus
212
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AND PASCHEN
after 8 min of ischemia (to 18.4 + 4.6 nmol/g/h, P c 0.05). Following 24 h of recirculation a significant reduction in enzyme activity was observed in the cortex after 8 min of ischemia (to 46.0 k 14.3 nmol/g/h, P < 0.05), in the striatum after 6 min (to 18.4 f 5.1 nmol/g/ h, P < 0.05), and in the hippocampus after 10 min (to 13.7 f 7.1 nmol/g/h, P < 0.05). Thus, the lowest enzyme activity after cerebral ischemia was found in the vulnerable hippocampus. The postischemic disturbances in polyamine synthesis are characterized by a sharp rise in ODC and decline in SAMDC activity (see above). These changes can therefore be illustrated best by the ratio of SAMDC/ ODC activity the degree of reduction of which is an indicator of the extent of ischemia-induced disturbances in polyamine synthesis. The ratio of SAMDC/ODC activity can be calculated from the results of the present study (Table 1). In control animals this ratio ranged between 54.3 + 17.2 and 172.4 f 54.7 indicating high SAMDC and low ODC activity. After only 4 min of ischemia and 8 h of recirculation the SAMDC/ODC ratio was sharply lowered in the hippocampus to 2.8 f 1.7 but was still 26.2 k 18.4, 39.9 + 50.5 and 39.8 + 58.1 in the cortex, striatum, and thalamus, respectively, thus illustrating that ischemia-induced disturbances in polyamine synthesis are most pronounced in the vulnerable hippocampus. DISCUSSION
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FIG. 2. SAMDC activity after 2-10 min of ischemia and 8 h (upper part) or 24 h (lower part) of recirculation. Data are expressed as nanomole per gram tissue per hour (means + SD). *P -z 0.05, **P < 0.01, ***P < 0.001, control, cf. experimental animals.
after 8 min of vascular occlusion. Following 24 h of recirculation ODC activity was close to control levels in all brain structures studied and this normalization was independent of the duration of ischemia. Ischemia-induced changes in SAMDC activity are summarized in Fig. 2. In control animals the highest enzyme activity was found in the cerebral cortex (69.9 2 10.0 nmol/g/h) while in the striatum, hippocampus, and thalamus enzyme activity was markedly lower and amounted to 27.1 + 5.3,23.1 f 3.4, and 26.9 + 5.4 nmol/ g/h. In the cerebral cortex a gradual decline in SAMDC activity was observed after 8 h of recirculation following 4 min (to 41.5 f 14.6 nmol/g/h, P < 0.01) to 10 min of ischemia (to 27.5 + 9.5 nmol/g/h, P < 0.001). In the striatum the activity was significantly decreased only
Ischemia-induced changes in the activity of both key enzymes in polyamine synthesis, ODC and SAMDC, have been studied previously by several groups (3,6,10, 17). In monkeys subjected to 1 h of complete cerebral ischemia the activity of these enzymes was sharply reduced immediately after the onset of recirculation, returned to control levels after about 1.5 h (ODC) or 11 h (SAMDC) of recirculation, and was markedly increased after periods of recirculation of up to 24 h (10). A similar reduction in enzyme activity has been observed after 30 min forebrain ischemia in rats during early recirculation (6). However, in rats in contrast to monkeys, SAMDC activity remained severely reduced throughout
TABLE
1
SAMDC/ODC Activity Ratio as Calculated from the Results Shown in Figures 1 and 2 after 2-10 min of Ischemia and 8 h of Recirculation Cortex Control 2 min 4 min 6 min 8 min 10 min Note.
172.4 213.5 26.2 25.0 8.4 9.7 Values
Striatum
+ 54.7 + 209.2 + 18.4 k 22.3 f 5.8 + 10.9 given
84.6 47.6 39.9 17.1 6.1 8.1
+ 37.8 k 34.6 + 50.5 f 8.7 + 1.7 f 5.1
are means
k SD.
Hippocampus 68.1 24.4 2.8 4.8 3.2 3.1
+ 28.0 rt 25.2 + 1.7 + 2.6 k 0.6 r 0.9
Thalamus 54.3 39.1 39.8 19.3 5.9 9.8
+ 17.2 k 34.5 + 58.1 + 9.8 + 1.9 + 5.4
ODC
AND
SAMDC
IN
TRANSIENT
the forebrain for up to 3 days after cerebral ischemia and ODC activity peaked at about 8 to 10 h of recirculation and declined thereafter (6). The results of the present series of experiments in which gerbils were used are similar to those obtained in rats: ODC activity was sharply increased after ischemia and 8 h of recirculation but returned to control levels after 24 h of reflow. SAMDC activity, in contrast, remained markedly reduced in the affected brain areas even 24 h after the onset of recirculation. It can be concluded, therefore, that ischemia-induced changes in polyamine metabolism vary in different animal species and that in small animals the suppression of SAMDC activity seems to be much more pronounced. The relationship between duration of cerebral ischemia and postischemic changes in ODC and SAMDC activity has, to the best of our knowledge, never been studied before. The results indicate that in the gerbil brain the response of ODC and SAMDC activity to transient cerebral ischemia is remarkably different: as indicated above, ODC activity was sharply increased during recirculation while SAMDC activity was reduced. Ischemia-induced changes in enzyme activities were only transient for ODC but permanent for SAMDC, at least during the first 24 h of recirculation. In addition, postischemic ODC activity reached a plateau when the duration of ischemia was extended up to 10 min while changes in SAMDC activity correlated with the duration of ischemia: the most pronounced decrease in SAMDC activity was observed in animals subjected to 10 min of ischemia. The observation that in the gerbil brain postischemic changes in ODC activity were only transient, peaking at about 8 to 10 h of recirculation while changes in SAMDC activity persisted for at least the first 24 h of reflow may indicate that the underlying molecular mechanisms are different for both enzymes. The half-lives for ODC and SAMDC amount to about 10 to 20 min and 60 min, respectively (20). Any change, therefore, in gene expression and protein synthesis affects ODC and SAMDC considerably faster and more markedly than proteins with a much longer half-life. Indeed, the gene expression and protein synthesis are markedly influenced by transient cerebral ischemia: when measured autoradiographically overall protein synthesis is almost completely suppressed during early recirculation (1,22, 24). Protein synthesis recovers almost to control values in nonvulnerable brain structures such as the cortex but remains severely suppressed in the most vulnerable region, the hippocampal CAl-subfield. In contrast, the gene expression of heat-shock proteins is sharply activated after cerebral ischemia (7, 9, 13). The postischemit changes in ODC and SAMDC activity may arise from these alterations: the reduction of SAMDC activity resulting from the suppression of overall protein synthesis and the increase in ODC activity resulting from the activation of stress-protein gene expression. The observations that the postischemic rise in ODC activity
CEREBRAL
ISCHEMIA
213
can be prevented by blocking protein synthesis (5), that ODC immunoreactivity is markedly enhanced in cortical neurons after cerebral ischemia (3), and that ODC mRNA levels are increased in the forebrain after cerebral ischemia (4) indicate that these changes are in fact produced by an activation of gene expression and not by allosteric mechanisms. The relationship between the ischemia-induced alteration of gene expression and changes in ODC and SAMDC activity is, however, not a perfect one. The postischemic reduction of SAMDC activity is much more prolonged than the suppression of protein synthesis, and ODC activity is considerably increased after cerebral ischemia even in the highly vulnerable hippocampal CAl-subfield (17) in which heatshock protein synthesis is not activated (13, 23). One prominent finding of the present series of experiments is the observation that following short-term ischemia the postischemic increase in ODC activity is closely related to the duration of ischemia and that the results from animals in which the period of ischemia was prolonged up to 10 min demonstrated that postischemit ODC activity had already reached a plateau, in the hippocampus after 4 min of ischemia and in the cortex, striatum, and thalamus after 8 min of vascular occlusion. It can be suggested, therefore, that the ischemiainduced activation of ODC gene expression is influenced by different parameters: first by a mechanism evoking a response which correlates with the duration of ischemia and second by a factor which prevents the tissue from reacting progressively to longer periods of ischemia with further increases in ODC activity. It is interesting to note that even after 30 min forebrain ischemia in rats (15) postischemic changes in ODC activity (determined by the identical analytical approach) were comparable to those observed in the present study after shorter, 6- to 8-min periods of ischemia. It has still to be established which factor is responsible for the ischemiainduced rise in ODC activity. Recently it has been hypothesized that calcium influx via the NMDA receptorgated ion channel plays a major role in the postischemic activation of ODC synthesis (11). Pharmacological interventions indicate, however, that the activation of ODC transcription and translation may be regulated by different mechanisms. The NMDA-receptor antagonist MK-801 prevented the postischemic increase in ODC activity (11) but did not influence the ischemia-induced activation of ODC gene expression (4). Barbiturate, in contrast, prevented the activation of ODC gene expression (4) but had no significant effect on the postischemit increase in ODC activity (17). The observation that postischemic ODC activity reached a plateau when the duration of ischemia was extended to 4 (hippocampus) or 8 min (cortex, striatum, and thalamus) as illustrated in the present study, whereas postischemic putrescine levels rose linearly with the duration of ischemia up to 30 min of vascular occlusion in rats (16), indicates that the increase in ODC activity is not the only cause for the overshoot in putrescine formation. After 30 min cerebral ischemia in rats postischemic pu-
214
RGHN,
SCHLENKER,
trescine levels were about five times as high as after 5 min ischemia in gerbils although the ischemia-induced changes in ODC activity were similar (see above and (15)). In fact, the postischemic suppression of SAMDC activity is much more pronounced when the duration of vascular occlusion is prolonged to 10 min (present study) or even to 30 min (6). It is, therefore, suggested that under conditions of prolonged transient vascular occlusion the reduction in SAMDC activity plays a major role in the postischemic overshoot in putrescine levels. The brain regions examined in the present study differ considerably in their response to ischemia, the hippocampus (particularly the CAl-subfield) being most vulnerable. It is interesting to note that the hippocampus reacted most sensitively to transient vascular occlusion. ODC activity was sharply activated after only 4 min of ischemia and within the whole forebrain the lowest SAMDC activity was observed in the hippocampus after 10 min of ischemia and 24 h of recirculation indicating a long-lasting effect of transient ischemia on SAMDC activity. A potentially important result of the present study is the observation that in the hippocampus after 4 min of ischemia (the threshold for production of neuronal damage in the hippocampal CAl-subfield) ODC activity shows considerable variance after 8 h of recirculation as illustrated by the huge standard deviation. In the cortex, striatum, and thalamus ODC activities varied between individual animals but values were evenly distributed between the lowest and highest levels. In contrast, two different groups could be identified in the hippocampus: four animals with ODC activity below 7 nmol/ g/h (range 3.6 to 6.9; mean + SD: 4.8 + 1.5 nmol/g/h) and three animals with ODC activity above 13 nmol/g/h (range 13.1 to 15.9; mean f SD: 14.5 + 1.4). A similar distribution of ODC activity values has been observed recently in our laboratory in a series of experiments in which the effects of barbiturate on postischemic changes in ODC activity were studied (17). In barbiturate-treated animals subjected to 5 min ischemia and 8 h of recirculation two different groups of animals could be identified: ODC activity values ranged in the first group of animals between 3.6 and 4.8 nmol/g/h (n = 3, mean +- SD: 4.1 -t 0.6 nmol/g/h) and in the second group of animals between 10.4 and 14.3 nmol/g/h (n = 4, mean k SD: 12.4 + 1.8 nmol/g/h). In the same series of experiments the ODC activity of untreated animals was below 7.0 nmol/g/h (n = 6). Thus, to establish a role for ODC in the pathological process of transient cerebral ischemia it would be of considerable interest to know the extent of cell damage developing at a later stage in animals in which the increase in ODC activity was most pronounced. ACKNOWLEDGMENT The excellent technical assistence of Claudia Kleppich is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft, Grant Pa 26613-2
AND PASCHEN REFERENCES 1. BODSCH, W., K. TAKAHASHI, A. BARBIER, B. GROSSE OPHOFF, AND K.-A. HOSSMANN. 1985. Cerebral protein synthesis andischemia. Prog. Brain Res. 63: 197-210. 2. BONDY, S. C., AND C. H. WALKER. 1986. Polyamines contribute to calcium-stimulated release of aspartate from brain particulate fractions. Bruin Res. 371: 96-100. 3. DEMPSEY, R. J., B. E. MALEY, D. COWEN, AND J. W. OLSON. 1988. Ornithine decarboxylase activity and immunohistochemical location in postischemic brain. J. Cereb. Blood Flow Metab. 8: 843-847. 4. DEMPSEY, R. J., J. M. CARNEY, AND M. S. KINDY. 1991. Modulation of ornithine decarboxylase mRNA following transient ischemia in the gerbil brain. J. Cereb. Blood Flow Metub. 11: 979985. 5. DIENEL, G. A., AND N. F. CRUZ. 1984. Induction of brain ornithine decarboxylase during recovery from metabolic, mechanical, thermal, or chemical injury. J. Neurochem. 42: 1053-1061. 6. DIENEL, G. A., N. F. CRUZ, AND S. J. ROSENFELD. 1985. Temporal profiles of protein responsive to transient ischemia. J. Neurochem. 44: 600-610. 7. DIENEL, G. A., M. KIEXSLING, M. JACEWICZ, AND W. A. PULSINELLI. 1986. Synthesis of heat shock proteins in rat brain cortex after transient ischemia. J. Cereb. Blood Flow Metub. 6: 505-510. 8. IQBAL, Z., AND H. KOENIG. 1985. Polyamines appear to be messengers in mediating Ca *+ fluxes and neurotransmitter release in potassium-depolarized synaptosomes. B&hem. Biophys. Res. Commun. 133: 563-573. 9. KIESSLING, M., G. A. DIENEL, M. JACEWICZ, AND W. A. PULSINELLI. 1986. Protein synthesis in postischemic rat brain: A twodimensional electrophoresis analysis. J. Cereb. Blood Flow Metab. 6: 642-649. 10. KLEIHUES, P., K.-A. HOSSMANN, A. E. PEGG, K. KOBAYASHI, AND V. ZIMMERMANN. 1975. Resuscitation of the monkey brain after one hour complete ischemia. III. Indications of metabolic recovery. Brain Res. 95: 61-73. 11. KOENIG, H., A. D. GOLDSTONE, C. Y. Lu, AND J. J. TROUT. 1990. Brain polyamines are controlled by N-methyl-D-aspartate receptors during ischemia and recirculation. Stroke Bl(supp1. III): 111-98-111-102. 12. KOMULAINEN, H., AND S. C. BONDY. 1987. Transient elevation of intrasynaptosomal free calcium by putrescine. Brain Res. 401: 50-54. 13. NOWAK, T. S. JR. 1990. Protein synthesis and the heat shock/ stress response after ischemia. Cerebrovasc. Brain Metab. Rev. 2: 345-366. 14. PASCHEN, W., R. SCHMIDT-KASTNER, B. DJURICIC, C. MEESE, F. LINN, AND K.-A. HOSSMANN. 1987. Polyamine changes in reversible cerebral ischemia. J. Neurochem. 49: 35-37. 15. PASCHEN, W., G. R~~HN, C. 0. MEESE, B. DJURICIC, AND R. SCHMIDT-KASTNER. 1988. Polyamine metabolism in reversible cerebral ischemia: Effect of o-difluoromethylornithine. Brain Res. 453: 9-16. 16. PASCHEN, W., R. SCHMIDT-KASTNER, J. HALLMAYER, AND B. DJURICIC. 1988. Polyamines in cerebral ischemia. Neurochem. Pathol. 9: l-20. 17. PASCHEN, W., J. HALLMAYER, G. MIES, AND G. R~HN. 1990. Ornithine decarboxylase activity and putrescine levels in reversible cerebral ischemia of Mongolian gerbils: Effect of barbiturate. J. Cereb. Blood Flow Metab. 10: 236-242. 18. PASCHEN, W., F. BENGTSSON, G. R~HN, P. BONNEKOH, B. SIESJ~, AND K.-A. HOSSMANN. 1991. Cerebral polyamine metabolism in reversible hypoglycemia of rat: Relationship to energy metabolites and calcium. J. Neurochem. 57: 204-215.
ODC AND SAMDC
IN TRANSIENT
19. PEGG, A. E., AND H. P&a. 1983. S-Adenosylmethionine decarboxylase (rat liver). In Methods in Enzymology (H. Tabor and C. W. Tabor, Eds.) Vol. 94, pp. 234-239. Academic Press, San Diego. 20. RUSSELL, D. H., AND S. H. SNYDER. 1969. Amine synthesis in regenerating rat liver: Extremely rapid turnover of ornithine decarboxylase. Mol. Pharmacol. 5: 253-262. 21. SEELY, J. E., AND A. E. PEGG. 1983. Ornithine decarboxylase (mouse kidney). In Methods in Enzymology (H. Tabor and C. W. Tabor, Eds.), Vol. 94, pp, 158-161. Academic Press, San Diego. 22. THILMANN, R., Y. XIE, P. KLEIHUES, AND M. KIESSLING. 1986. Persistent inhibition of protein synthesis precedes delayed neu-
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23. VASS, K., S. TOMIDA, K.-A. HOSSMANN, T. S. NOWAK, JR., AND I. KLATZO. 1988. Microvascular disturbances and edema formation after repetitive ischemia of gerbil brain. A& Neuropathol. (Berlin)
75: 288-294.
24. XIE, Y., K.-A. HOSSMANN, K. MUNEKATA, AND K. SEO. 1987. Prolonged suppression of protein synthesis after brief cerebral ischemia in gerbils. In Stroke and Microcirculation (J. CervosNavarro and R. Ferszt, Eds.), pp. 135-141. Raven Press, New York.
NEUROLOGY
117,210-215
(1992)
Activity of Ornithine Decarboxylase and S-Adenosylmethionine Decarboxylase in Transient Cerebral Ischemia: Relationship to the Duration of Vascular Occlusion GABRIELERGHN,MICHAELSCHLENKER,ANDWULFPASCHEN Max-Planck
Institute
for Neurological
Research,
Department
Copyright All rights
Inc. reserved.
cologne,
Germany
underlyand pro-
INTRODUCTION The synthesis of the polyamines spermidine and spermine and their precursor putrescine is controlled by the activity of the two key enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). Transient cerebral ischemia induces marked changes in polyamine synthesis, namely a sharp increase in ODC and decrease in SAMDC activity (3,6,10, 15). The result is an overshoot in the formation of putrescine, the product of the ODC reaction, because synthesis of putrescine is activated whereas the interconversion of putrescine into spermidine and spermine is suppressed due to low SAMDC activity. Since postischemit putrescine levels correlate closely with the density of neuronal necrosis (15) and putrescine mediates neurotransmitter release from nerve endings and calcium fluxes at the cell membrane (2, 8, 12) it has been suggested that this compound is involved in the manifestation of ischemia- or hypoglycemia-induced neuronal necrosis (14,18). It has been shown previously that during recirculation putrescine levels correlate closely with the duration of ischemia (16), but no information has been available up to now to indicate whether any such relationship exists between the duration of ischemia and the postischemic ODC and SAMDC activity. In the present series of experiments, therefore, postischemic changes in ODC and SAMDC activity were studied in relation to the duration of ischemia. Animals were subjected to 2,4,6,8, or 10 min of ischemia and 8 or 24 h of recovery. A threshold ischemic duration for ODC induction was observed, the duration of ischemia needed to produce a significant increase in ODC activity being 4 min in the hippocampus, 6 min in the cortex and striaturn, and 8 min in the thalamus. 210
$5.00
0 1992 by Academic Press, of reproduction in any form
Neurology,
information about the molecular mechanisms ing stress-induced changes in gene expression tein synthesis. o 1992 Academic PWSS, h.
Mongolian gerbils were anesthetized with halothane and forebrain ischemia was induced by occluding both common carotid arteries. After 2,4,6,8, or 10 min of vascular occlusion clips were removed and animals allowed to recover for 8 or 24 h. At the end of the experiments animals were reanesthetized and their brains frozen in situ. Tissue samples were taken from the cerebral cortex, striatum, hippocampus, and thalamus for determination of ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (SAMDC) activity by measurement of the release of 14C0, from [14C]ornithine and 5’-[‘4C]adenosylmethionine, respectively. A transient increase in ODC activity was found after 8 h of recirculation following cerebral ischemia in all brain structures studied. ODC activity was significantly increased after 8 h of recirculation in the hippocampus of animals subjected to 4 min of ischemia, in the cortex and striatum after 6 min of ischemia, and in the thalamus after 8 min of vascular occlusion. ODC activity had already reached a plateau in the hippocampus after 4 min of vascular occlusion and in the cortex, striatum, and thalamus after 8 min, since there is no further increase in activity even after 10 min of ischemia. After cerebral ischemia and 24 h of recirculation ODC activity returned to control levels throughout the forebrain regardless of the duration of ischemia. SAMDC activity was significantly reduced after 8 h of recirculation following 4 to 10 min of ischemia in the cortex and 8 min of ischemia in the striatum. After 24 h of recirculation a significant reduction of SAMDC activity was measured in the cortex of animals subjected to 8 or 10 min of ischemia, in the striatum following 6 or 10 min of ischemia, and after 10 min of ischemia in the hippocampus. In control animals the SAMDUODC activity ratio (as an index of the extent of the disturbance of polyamine synthesis) ranged between 54.3 f 17.2 (thalamus) and 172.4 + 54.7 (cortex). After only 4 min of ischemia and 8 h of recirculation this ratio declined to 2.8 + 1.7 in the vulnerable hippocampus but was still between 26.2 + 18.4 (cortex) and 39.9 + 50.5 (striaturn). It is suggested that studying ischemia-induced changes in ODC and SAMDC activity may provide new 0014-4666192
of Experimental
ODC
MATERIALS
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SAMDC
IN
TRANSIENT
CEREBRAL
211
ISCHEMIA
METHODS
8h recirculation
Animal Experiments
Striatum
Sixty-six male Mongolian gerbils (Meriones unguiculatus) weighing 50-80 g were used. Both common carotid arteries were occluded under anesthesia (1.5% halothane in 70% N,O and 30% 0,) with aneurysm clips for 2,4,6,8, or 10 min. During anesthesia rectal temperature was measured and maintained at 375°C by a feedback-controlled infrared heating system. Sham-operated animals in which both carotid arteries were exposed but not occluded served as controls. At the end of ischemia the clips were removed to allow reperfusion and the skin incision was sutured. After 8 or 24 h of recirculation the animals were reanesthetized and the brains frozen in situ with liquid nitrogen. Brains were removed from the skull at -20°C. Tissue samples were taken from the cerebral cortex, the striatum, the hippocampus, and the thalamus for measurement of enzyme activities.
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All data are expressed as means f SD. Statistical analysis was performed using Student’s t test with the Bonferroni corrections for multiple comparisons. A P value of less than 0.05 was considered to indicate significant differences. RESULTS
The activities of ODC measured after 2 to 10 min of ischemia and 8 or 24 h of recirculation are shown in Fig. 1. In the brains of control animals no regional differences in ODC activity could be detected: average enzyme activity amounted to 0.43 f 0.11, 0.36 + 0.11, 0.40
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Enzyme Analysis Determination of the activity of ornithine decarboxylase (ODC, E.C. 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMDC, E.C. 4.1.1.50) was performed as described elsewhere (19, 21) with some modifications (15). In brief, brain samples were homogenized in 25 vols (w/v) Tris/HCl buffer (50 mM, pH 7.2, supplemented with 5 mM DTT and 0.1 mM EGTA). The test mix for the measurement of ODC activity was composed of brain homogenate (4 mg of tissue), pyridoxal-5-phosphate (54 PLM), and L-[1-14C]ornithine (74 pLM, 51.6 mCi/mmol, New England Nuclear Corporation) in a total volume of 130 ~1. For the determination of SAMDC activity brain homogenate (1 mg of tissue), putrescine (2.5 mM), EDTA (1 n&Q and S-[carboxy-14C]adenosylL-methionine (0.2 mM, 16.7 mCi/mmol, New England Nuclear Corporation) were mixed yielding a final volume of 140 ~1. Enzyme activities were quantified by measuring the release of 14C0, from [14C]ornithine and S-[14C]adenosylmethionine for ODC and SAMDC, respectively.
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FIG. 1. ODC activity after 2-10 min of ischemia and 8 h (upper part) or 24 h (lower part) of recirculation. Data are expressed as nanomoles per gram tissue per hour (means t SD). **P < 0.01, ***P < 0.001, control, cf. experimental animals.
i 0.18,0.52 +- 0.11 nmol/g/h in the cortex, striatum hippocampus, and thalamus, respectively. After 8 h of recirculation there was a significant increase in enzyme activity; the temporal profile varied however in different brain structures. Postischemic ODC activity was significantly increased in the hippocampus after only 4 min of ischemia (to 9.01 f 5.36 nmol/g/h, P < 0.01, cf. controls), in the cortex and striatum after 6 min of vascular occlusion (to 3.16 + 1.83 and 2.00 -t 1.12 nmol/g/h, respectively, P < 0.01, cf. controls), and in the thalamus after 8 min of ischemia (to 4.07 + 0.63 nmol/g/h, P -C 0.001, cf. controls). When the duration of ischemia was extended ODC activity was shown to have reached a plateau, in the hippocampus already after 4 min of ischemia and in the cerebral cortex, striatum, and thalamus
212
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AND PASCHEN
after 8 min of ischemia (to 18.4 + 4.6 nmol/g/h, P c 0.05). Following 24 h of recirculation a significant reduction in enzyme activity was observed in the cortex after 8 min of ischemia (to 46.0 k 14.3 nmol/g/h, P < 0.05), in the striatum after 6 min (to 18.4 f 5.1 nmol/g/ h, P < 0.05), and in the hippocampus after 10 min (to 13.7 f 7.1 nmol/g/h, P < 0.05). Thus, the lowest enzyme activity after cerebral ischemia was found in the vulnerable hippocampus. The postischemic disturbances in polyamine synthesis are characterized by a sharp rise in ODC and decline in SAMDC activity (see above). These changes can therefore be illustrated best by the ratio of SAMDC/ ODC activity the degree of reduction of which is an indicator of the extent of ischemia-induced disturbances in polyamine synthesis. The ratio of SAMDC/ODC activity can be calculated from the results of the present study (Table 1). In control animals this ratio ranged between 54.3 + 17.2 and 172.4 f 54.7 indicating high SAMDC and low ODC activity. After only 4 min of ischemia and 8 h of recirculation the SAMDC/ODC ratio was sharply lowered in the hippocampus to 2.8 f 1.7 but was still 26.2 k 18.4, 39.9 + 50.5 and 39.8 + 58.1 in the cortex, striatum, and thalamus, respectively, thus illustrating that ischemia-induced disturbances in polyamine synthesis are most pronounced in the vulnerable hippocampus. DISCUSSION
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FIG. 2. SAMDC activity after 2-10 min of ischemia and 8 h (upper part) or 24 h (lower part) of recirculation. Data are expressed as nanomole per gram tissue per hour (means + SD). *P -z 0.05, **P < 0.01, ***P < 0.001, control, cf. experimental animals.
after 8 min of vascular occlusion. Following 24 h of recirculation ODC activity was close to control levels in all brain structures studied and this normalization was independent of the duration of ischemia. Ischemia-induced changes in SAMDC activity are summarized in Fig. 2. In control animals the highest enzyme activity was found in the cerebral cortex (69.9 2 10.0 nmol/g/h) while in the striatum, hippocampus, and thalamus enzyme activity was markedly lower and amounted to 27.1 + 5.3,23.1 f 3.4, and 26.9 + 5.4 nmol/ g/h. In the cerebral cortex a gradual decline in SAMDC activity was observed after 8 h of recirculation following 4 min (to 41.5 f 14.6 nmol/g/h, P < 0.01) to 10 min of ischemia (to 27.5 + 9.5 nmol/g/h, P < 0.001). In the striatum the activity was significantly decreased only
Ischemia-induced changes in the activity of both key enzymes in polyamine synthesis, ODC and SAMDC, have been studied previously by several groups (3,6,10, 17). In monkeys subjected to 1 h of complete cerebral ischemia the activity of these enzymes was sharply reduced immediately after the onset of recirculation, returned to control levels after about 1.5 h (ODC) or 11 h (SAMDC) of recirculation, and was markedly increased after periods of recirculation of up to 24 h (10). A similar reduction in enzyme activity has been observed after 30 min forebrain ischemia in rats during early recirculation (6). However, in rats in contrast to monkeys, SAMDC activity remained severely reduced throughout
TABLE
1
SAMDC/ODC Activity Ratio as Calculated from the Results Shown in Figures 1 and 2 after 2-10 min of Ischemia and 8 h of Recirculation Cortex Control 2 min 4 min 6 min 8 min 10 min Note.
172.4 213.5 26.2 25.0 8.4 9.7 Values
Striatum
+ 54.7 + 209.2 + 18.4 k 22.3 f 5.8 + 10.9 given
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+ 37.8 k 34.6 + 50.5 f 8.7 + 1.7 f 5.1
are means
k SD.
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+ 28.0 rt 25.2 + 1.7 + 2.6 k 0.6 r 0.9
Thalamus 54.3 39.1 39.8 19.3 5.9 9.8
+ 17.2 k 34.5 + 58.1 + 9.8 + 1.9 + 5.4
ODC
AND
SAMDC
IN
TRANSIENT
the forebrain for up to 3 days after cerebral ischemia and ODC activity peaked at about 8 to 10 h of recirculation and declined thereafter (6). The results of the present series of experiments in which gerbils were used are similar to those obtained in rats: ODC activity was sharply increased after ischemia and 8 h of recirculation but returned to control levels after 24 h of reflow. SAMDC activity, in contrast, remained markedly reduced in the affected brain areas even 24 h after the onset of recirculation. It can be concluded, therefore, that ischemia-induced changes in polyamine metabolism vary in different animal species and that in small animals the suppression of SAMDC activity seems to be much more pronounced. The relationship between duration of cerebral ischemia and postischemic changes in ODC and SAMDC activity has, to the best of our knowledge, never been studied before. The results indicate that in the gerbil brain the response of ODC and SAMDC activity to transient cerebral ischemia is remarkably different: as indicated above, ODC activity was sharply increased during recirculation while SAMDC activity was reduced. Ischemia-induced changes in enzyme activities were only transient for ODC but permanent for SAMDC, at least during the first 24 h of recirculation. In addition, postischemic ODC activity reached a plateau when the duration of ischemia was extended up to 10 min while changes in SAMDC activity correlated with the duration of ischemia: the most pronounced decrease in SAMDC activity was observed in animals subjected to 10 min of ischemia. The observation that in the gerbil brain postischemic changes in ODC activity were only transient, peaking at about 8 to 10 h of recirculation while changes in SAMDC activity persisted for at least the first 24 h of reflow may indicate that the underlying molecular mechanisms are different for both enzymes. The half-lives for ODC and SAMDC amount to about 10 to 20 min and 60 min, respectively (20). Any change, therefore, in gene expression and protein synthesis affects ODC and SAMDC considerably faster and more markedly than proteins with a much longer half-life. Indeed, the gene expression and protein synthesis are markedly influenced by transient cerebral ischemia: when measured autoradiographically overall protein synthesis is almost completely suppressed during early recirculation (1,22, 24). Protein synthesis recovers almost to control values in nonvulnerable brain structures such as the cortex but remains severely suppressed in the most vulnerable region, the hippocampal CAl-subfield. In contrast, the gene expression of heat-shock proteins is sharply activated after cerebral ischemia (7, 9, 13). The postischemit changes in ODC and SAMDC activity may arise from these alterations: the reduction of SAMDC activity resulting from the suppression of overall protein synthesis and the increase in ODC activity resulting from the activation of stress-protein gene expression. The observations that the postischemic rise in ODC activity
CEREBRAL
ISCHEMIA
213
can be prevented by blocking protein synthesis (5), that ODC immunoreactivity is markedly enhanced in cortical neurons after cerebral ischemia (3), and that ODC mRNA levels are increased in the forebrain after cerebral ischemia (4) indicate that these changes are in fact produced by an activation of gene expression and not by allosteric mechanisms. The relationship between the ischemia-induced alteration of gene expression and changes in ODC and SAMDC activity is, however, not a perfect one. The postischemic reduction of SAMDC activity is much more prolonged than the suppression of protein synthesis, and ODC activity is considerably increased after cerebral ischemia even in the highly vulnerable hippocampal CAl-subfield (17) in which heatshock protein synthesis is not activated (13, 23). One prominent finding of the present series of experiments is the observation that following short-term ischemia the postischemic increase in ODC activity is closely related to the duration of ischemia and that the results from animals in which the period of ischemia was prolonged up to 10 min demonstrated that postischemit ODC activity had already reached a plateau, in the hippocampus after 4 min of ischemia and in the cortex, striatum, and thalamus after 8 min of vascular occlusion. It can be suggested, therefore, that the ischemiainduced activation of ODC gene expression is influenced by different parameters: first by a mechanism evoking a response which correlates with the duration of ischemia and second by a factor which prevents the tissue from reacting progressively to longer periods of ischemia with further increases in ODC activity. It is interesting to note that even after 30 min forebrain ischemia in rats (15) postischemic changes in ODC activity (determined by the identical analytical approach) were comparable to those observed in the present study after shorter, 6- to 8-min periods of ischemia. It has still to be established which factor is responsible for the ischemiainduced rise in ODC activity. Recently it has been hypothesized that calcium influx via the NMDA receptorgated ion channel plays a major role in the postischemic activation of ODC synthesis (11). Pharmacological interventions indicate, however, that the activation of ODC transcription and translation may be regulated by different mechanisms. The NMDA-receptor antagonist MK-801 prevented the postischemic increase in ODC activity (11) but did not influence the ischemia-induced activation of ODC gene expression (4). Barbiturate, in contrast, prevented the activation of ODC gene expression (4) but had no significant effect on the postischemit increase in ODC activity (17). The observation that postischemic ODC activity reached a plateau when the duration of ischemia was extended to 4 (hippocampus) or 8 min (cortex, striatum, and thalamus) as illustrated in the present study, whereas postischemic putrescine levels rose linearly with the duration of ischemia up to 30 min of vascular occlusion in rats (16), indicates that the increase in ODC activity is not the only cause for the overshoot in putrescine formation. After 30 min cerebral ischemia in rats postischemic pu-
214
RGHN,
SCHLENKER,
trescine levels were about five times as high as after 5 min ischemia in gerbils although the ischemia-induced changes in ODC activity were similar (see above and (15)). In fact, the postischemic suppression of SAMDC activity is much more pronounced when the duration of vascular occlusion is prolonged to 10 min (present study) or even to 30 min (6). It is, therefore, suggested that under conditions of prolonged transient vascular occlusion the reduction in SAMDC activity plays a major role in the postischemic overshoot in putrescine levels. The brain regions examined in the present study differ considerably in their response to ischemia, the hippocampus (particularly the CAl-subfield) being most vulnerable. It is interesting to note that the hippocampus reacted most sensitively to transient vascular occlusion. ODC activity was sharply activated after only 4 min of ischemia and within the whole forebrain the lowest SAMDC activity was observed in the hippocampus after 10 min of ischemia and 24 h of recirculation indicating a long-lasting effect of transient ischemia on SAMDC activity. A potentially important result of the present study is the observation that in the hippocampus after 4 min of ischemia (the threshold for production of neuronal damage in the hippocampal CAl-subfield) ODC activity shows considerable variance after 8 h of recirculation as illustrated by the huge standard deviation. In the cortex, striatum, and thalamus ODC activities varied between individual animals but values were evenly distributed between the lowest and highest levels. In contrast, two different groups could be identified in the hippocampus: four animals with ODC activity below 7 nmol/ g/h (range 3.6 to 6.9; mean + SD: 4.8 + 1.5 nmol/g/h) and three animals with ODC activity above 13 nmol/g/h (range 13.1 to 15.9; mean f SD: 14.5 + 1.4). A similar distribution of ODC activity values has been observed recently in our laboratory in a series of experiments in which the effects of barbiturate on postischemic changes in ODC activity were studied (17). In barbiturate-treated animals subjected to 5 min ischemia and 8 h of recirculation two different groups of animals could be identified: ODC activity values ranged in the first group of animals between 3.6 and 4.8 nmol/g/h (n = 3, mean +- SD: 4.1 -t 0.6 nmol/g/h) and in the second group of animals between 10.4 and 14.3 nmol/g/h (n = 4, mean k SD: 12.4 + 1.8 nmol/g/h). In the same series of experiments the ODC activity of untreated animals was below 7.0 nmol/g/h (n = 6). Thus, to establish a role for ODC in the pathological process of transient cerebral ischemia it would be of considerable interest to know the extent of cell damage developing at a later stage in animals in which the increase in ODC activity was most pronounced. ACKNOWLEDGMENT The excellent technical assistence of Claudia Kleppich is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft, Grant Pa 26613-2
AND PASCHEN REFERENCES 1. BODSCH, W., K. TAKAHASHI, A. BARBIER, B. GROSSE OPHOFF, AND K.-A. HOSSMANN. 1985. Cerebral protein synthesis andischemia. Prog. Brain Res. 63: 197-210. 2. BONDY, S. C., AND C. H. WALKER. 1986. Polyamines contribute to calcium-stimulated release of aspartate from brain particulate fractions. Bruin Res. 371: 96-100. 3. DEMPSEY, R. J., B. E. MALEY, D. COWEN, AND J. W. OLSON. 1988. Ornithine decarboxylase activity and immunohistochemical location in postischemic brain. J. Cereb. Blood Flow Metab. 8: 843-847. 4. DEMPSEY, R. J., J. M. CARNEY, AND M. S. KINDY. 1991. Modulation of ornithine decarboxylase mRNA following transient ischemia in the gerbil brain. J. Cereb. Blood Flow Metub. 11: 979985. 5. DIENEL, G. A., AND N. F. CRUZ. 1984. Induction of brain ornithine decarboxylase during recovery from metabolic, mechanical, thermal, or chemical injury. J. Neurochem. 42: 1053-1061. 6. DIENEL, G. A., N. F. CRUZ, AND S. J. ROSENFELD. 1985. Temporal profiles of protein responsive to transient ischemia. J. Neurochem. 44: 600-610. 7. DIENEL, G. A., M. KIEXSLING, M. JACEWICZ, AND W. A. PULSINELLI. 1986. Synthesis of heat shock proteins in rat brain cortex after transient ischemia. J. Cereb. Blood Flow Metub. 6: 505-510. 8. IQBAL, Z., AND H. KOENIG. 1985. Polyamines appear to be messengers in mediating Ca *+ fluxes and neurotransmitter release in potassium-depolarized synaptosomes. B&hem. Biophys. Res. Commun. 133: 563-573. 9. KIESSLING, M., G. A. DIENEL, M. JACEWICZ, AND W. A. PULSINELLI. 1986. Protein synthesis in postischemic rat brain: A twodimensional electrophoresis analysis. J. Cereb. Blood Flow Metab. 6: 642-649. 10. KLEIHUES, P., K.-A. HOSSMANN, A. E. PEGG, K. KOBAYASHI, AND V. ZIMMERMANN. 1975. Resuscitation of the monkey brain after one hour complete ischemia. III. Indications of metabolic recovery. Brain Res. 95: 61-73. 11. KOENIG, H., A. D. GOLDSTONE, C. Y. Lu, AND J. J. TROUT. 1990. Brain polyamines are controlled by N-methyl-D-aspartate receptors during ischemia and recirculation. Stroke Bl(supp1. III): 111-98-111-102. 12. KOMULAINEN, H., AND S. C. BONDY. 1987. Transient elevation of intrasynaptosomal free calcium by putrescine. Brain Res. 401: 50-54. 13. NOWAK, T. S. JR. 1990. Protein synthesis and the heat shock/ stress response after ischemia. Cerebrovasc. Brain Metab. Rev. 2: 345-366. 14. PASCHEN, W., R. SCHMIDT-KASTNER, B. DJURICIC, C. MEESE, F. LINN, AND K.-A. HOSSMANN. 1987. Polyamine changes in reversible cerebral ischemia. J. Neurochem. 49: 35-37. 15. PASCHEN, W., G. R~~HN, C. 0. MEESE, B. DJURICIC, AND R. SCHMIDT-KASTNER. 1988. Polyamine metabolism in reversible cerebral ischemia: Effect of o-difluoromethylornithine. Brain Res. 453: 9-16. 16. PASCHEN, W., R. SCHMIDT-KASTNER, J. HALLMAYER, AND B. DJURICIC. 1988. Polyamines in cerebral ischemia. Neurochem. Pathol. 9: l-20. 17. PASCHEN, W., J. HALLMAYER, G. MIES, AND G. R~HN. 1990. Ornithine decarboxylase activity and putrescine levels in reversible cerebral ischemia of Mongolian gerbils: Effect of barbiturate. J. Cereb. Blood Flow Metab. 10: 236-242. 18. PASCHEN, W., F. BENGTSSON, G. R~HN, P. BONNEKOH, B. SIESJ~, AND K.-A. HOSSMANN. 1991. Cerebral polyamine metabolism in reversible hypoglycemia of rat: Relationship to energy metabolites and calcium. J. Neurochem. 57: 204-215.
ODC AND SAMDC
IN TRANSIENT
19. PEGG, A. E., AND H. P&a. 1983. S-Adenosylmethionine decarboxylase (rat liver). In Methods in Enzymology (H. Tabor and C. W. Tabor, Eds.) Vol. 94, pp. 234-239. Academic Press, San Diego. 20. RUSSELL, D. H., AND S. H. SNYDER. 1969. Amine synthesis in regenerating rat liver: Extremely rapid turnover of ornithine decarboxylase. Mol. Pharmacol. 5: 253-262. 21. SEELY, J. E., AND A. E. PEGG. 1983. Ornithine decarboxylase (mouse kidney). In Methods in Enzymology (H. Tabor and C. W. Tabor, Eds.), Vol. 94, pp, 158-161. Academic Press, San Diego. 22. THILMANN, R., Y. XIE, P. KLEIHUES, AND M. KIESSLING. 1986. Persistent inhibition of protein synthesis precedes delayed neu-
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24. XIE, Y., K.-A. HOSSMANN, K. MUNEKATA, AND K. SEO. 1987. Prolonged suppression of protein synthesis after brief cerebral ischemia in gerbils. In Stroke and Microcirculation (J. CervosNavarro and R. Ferszt, Eds.), pp. 135-141. Raven Press, New York.
