Journal of Neurochemisrry Raven Press, Ltd., New York 0 1992 International Society for Neurochemistry
Transcription-Dependent and -Independent Induction of Cerebral Ornithine Decarboxylase N.H. Zawia and S. C. Bondy Departments of Pharmacology and Community and Environmental Medicine and The Southern Occupational Health Center, University of CalSfornia,Irvine, California, U.S.A.
Abstract: Ornithine decarboxylase ( O X ; EC 4.I. 1.17) is a highly inducible, rate-limiting enzyme of the polyamine pathway. We have studied the mechanisms that lead to the induction of ODC activity in response to electrical stimulation in three brain regions. Hippocampal ODC activity was found to exhibit much larger elevations than that of the neocortex and the cerebellum. The levels of ODC gene expression were also followed to examine its relationship to the existing regional differences in ODC activity. In the neocortex, there was an elevation of both the ODC mRNA and enzyme activity. However, the hippocampal ODC mRNA level was not increased by electroconvulsive shock. Furthermore, the effects of hormonaI changes and seizures on these regional differ-
ences in ODC induction were also examined. Adrenalectomy did not affect ODC activity, but pretreatment with the anticonvulsant MK-801 caused a depression of the induced levels of enzyme activity. Our data suggest that ODC activity in all the brain regions studied is directly elevated by electrically stimulated seizures. However, this induced ODC activity may or may not involve enhanced gene expression. Key Words: Ornithine decarboxylase-mRNA-Electrical stimulation-Neocortex-Hippocampus-Induction. Zawia N. H. and Bondy S. C. Transcription-dependent and -independent induction of cerebral ornithine decarboxylase. J. Neurochem. 58, 736-739 (1992).
Induction of the enzyme ornithine decarboxylase (ODC; EC 4.1.1.17) is one of the earliest events that occur following perturbations to neural tissue (Koenig et al., 1983; Dornay et al., 1986; Wells, 1987; Gilad and Gilad, 1988). The elevation of ODC activity, following tissue damage, results in increased polyamine levels, which are believed to play a key role in the adaptive mechanisms of the CNS (Dienel and Cruz, 1984; Agnati et al., 1985; Kanje et al., 1986; Slotkin and Bartolome, 1986). The adult brain has a very low level of ODC activity basally, but this enzyme is highly inducible. Among the factors that play a significant role in the regulation of ODC activity are mRNA transcription and the turnover rate of the ODC protein (McCann, 1980). Induction of cerebral ODC activity by electrical stimulation has been reported (Pajunen et al., 1978). The hippocampus exhibits the greatest increases in ODC enzyme activity, in response to electroconvulsive shock (ECS), followed by the neocortex and the cerebellum (Bondy et al., 1987). However, it was not clear whether these varying degrees of ODC induction are
the outcome of the same process, or whether they reflect inherently different regional modes of ODC regulation. Recently we have shown that the induction of neocortical ODC activity by ECS is accompanied by a rapid elevation in the levels of the ODC mRNA (Zawia and Bondy, 1990). Here, we examine the regulation of ODC activity in response to electrical stimulation in three brain regions: neocortex, hippocampus, and cerebellum. At the molecular level, the rise in ODC activity has been compared with changes in the levels of the ODC mRNA. Furthermore, some factors potentially responsible for the fluctuations in ODC activity in each brain region have also been investigated. To this end, the modulation of the stimulated regional ODC activity by adrenalectomy or pretreatment with the anticonvulsant (+)-5-methyl-l0,1 l-dihydro-5H-dibenzo[u,d]cyclohepten-5,lO-imine (MK-80 1) were examined.
Received March 6 , 199I ; accepted July 17, I99 1. Address correspondence and reprint requests to Dr. N. H. Zawia at his present address: National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A.
Abbreviations used ECS, electroconvulsive shock; MK-801, (+)5-methyl-l0,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine; O K , ornithine decarboxylase.
MATERIALS AND METHODS Animals and treatment Male Fischer CD rats, 6-10 weeks old, were used. The animals were housed at 22°C with free access to food and
736
DIFFERENTIAL REGULATION OF CEREBRAL ODC water, on a 12-h light-dark cycle. ECS was administered via ear electrodes, with saline-moistened contact pads, using a constant-current apparatus. Shock was applied to normal, adrenalectomized (Zivic-Miller, Zelinople, PA, U.S.A.), and sham-operated (surgery performed but no adrenal glands removed) rats at the following intensity: 85 mA for 1 s with 1ms pulses at a frequency of 50 Hz. Control animals were treated similarly without the application of a current. These shock intensities have been demonstrated to be without detectable morphological damage to the brain (Dam, 1982). Some animals were dosed by intraperitoneal injections of the agent MK-80 1 (1 mg/kg), dissolved in saline. This agent is a selective antagonist of the N-methyl-D-aspartate-type of glutamate receptor (Wong et al., 1986). Animals were decapitated, and their corresponding brain regions were dissected out, quickly placed in dry ice, and kept at -70°C until use.
737
T
Neocortex
Hippocampus
Cerebellum
FIG. 1. Activities of ODC in different brain regions 5 h after ECS. Data are mean f SE (bars) values from seven to nine individual
determinations. Values significantly different from corresponding basal values using a two-tailed Student's t test are indicated: ' p < 0.001.
ODC assay ODC activity was determined by measurement of evolved I4CO2from carboxy [14C]ornithine(55.9 mCi/nmol; New England Nuclear, Boston, MA, U.S.A.). Tissue was homogenized in 19 volumes of 0.04 M Tns-HCI. After centrifugation (26,000 g,20 min), 0.9 ml of various tissue preparations was added to 50 pl of pyridoxal phosphate solution (1 nM) and 50 p1 of [I4C]ornithine, in the presence of 0.045 M dithiothreitol. The final ornithine concentration was 2.5 FM. Incubations were carried out at 37°C for 45 min in a sealed tube and terminated by injection of 1 ml of 2 M acetic acid into the reaction mixture (Russell and Snyder, 1969).Evolved I4CO2was trapped on a paper wick containing hyamine suspended above the reaction mixture. The decarboxylation process is linear for up to 1.5 h under these conditions. Decarboxylation not attributable to ODC was determined by assaying a parallel incubation in the presence of 5 W d i f l u oromethylornithine, a specific inhibitor of ODC (Metcalf et al., 1978).
RNA analysis Total cellular RNA was isolated from brain regions of control and ECS-treated animals using the phenol-sodium dodecyl sulfate method (Maniatis et al., 1982;Soreq et al., 1983). The RNA was fractionated according to size in formaldehydecontaining agarose gels (Fourney et al., 1988) and transferred onto nitrocellulose filter paper. The filters were then probed with a nick-translation 32P-labeled ODC cDNA plasrnid (McConlogue et al., 1984), washed under stringent conditions, and exposed to x-ray film at -70°C for 5-7 days (Thomas, 1980). The mRNA content was estimated by scanning the autoradiograms with a laser densitometer (LKB, Pharmacia), interfaced with a computer integrator. The areas under each peak were integrated to provide a quantitative estimate of the levels of the ODC mRNA.
RESULTS ODC activity Basal ODC activity in the hippocampus is twice as high as that in the neocortex and much higher than that in the cerebellum (Fig. I). ECS resulted in regional differences in the induction of ODC activity in the brain. The highest elevation in activity was seen in the hippocampus (fivefold), followed by the neocortex (twofold), whereas the cerebellum exhibited the least
stimulation of ODC activity (1.5-fold). These enzyme assays of ODC activity from different brain regions were consistent with earlier reports (Bondy et al., 1987).
ODC mRNA The basal ODC mRNA levels were similar in all brain regions examined (Fig. 2). ECS also produced regional differences in the induction of the ODC mRNA (Fig. 2). In contrast to the dramatic induction of enzyme activity in the hippocampus, the ODC mRNA levels did not change significantly in this brain region following ECS (Fig. 2). There was also no change in the level of the ODC message in the cerebellum. The neocortex, on the other hand, as previously shown, exhibited a significant elevation in the levels of ODC mRNA following ECS (Zawia and Bondy, 1990). Adrenalectomy and MK-801 Adrenalectomy had no significant effect on ECS-induced enzyme activity in any of the brain regions studied (Fig. 3). On the other hand, MK-80 1 pretreatment, which resulted in the abolition of the tonic-clonic seizures produced by ECS, effected a 40% attenuation of the ECS-induced ODC activity in all the areas surveyed (Fig. 4). Therefore, the induction of ODC was directly related to seizure activity.
DISCUSSION It is tempting to generalize that the regulation of an enzyme would be similar in all regions of the same organ. However, such an assumption is not true for cerebral ODC. The basal ODC activities exhibit some regional specificity, whereas the levels of the ODC mRNA appear to be similar in all brain regions examined (Figs. 1 and 2). Because the genomic capacity for ODC synthesis in all the brain regions appears to be the same, this suggests that some additional cytoplasmic components are involved in varying these regional levels of ODC activity, especially in maintaining the relatively high basal levels in the hippocampus. J. Neurochem.. Vol. 58. No. 2, 1992
N . H. ZAWIA A N D S. C. BONDY
738
T 0
BasalLevel ECS only
m
NEOCORTEX
0
BasalmRNA InducedmRNA
T T
NEOCORTEX
HIPPOCAMPUS
CEREBELLUM
Autoradiograms exhibiting ODC mRNA bands were scanned, and the intensity of the bands per integrated area was used to quantify the results. The autoradiograms shown are from two separate representative determinations.The autoradiograms with the basal (b) and ECS-induced (ecs) hippocampal ODC mRNA levels were exposed for a longer time to enhance the detection of the otherwise low ODC mRNA levels. In this blot, a lane of the basal neocortex (ntx) levels was included to show that the basal ODC mRNA levels in the two regions are similar. Data are mean It_ SE (bars) values from four to six individual determinations. Each determinationof hippocampal mRNA levels consisted of pooled tissue from several animals. Values significantly different from basal values using a two-tailed Student's t test are indicated: ' p < 0.05.
z "1
HIPPOCAMPUS
CEREBELLUM
FIG. 3. Effect of adrenalectomy on ECS-induced ODC activity in
various brain regions. To avoid the inclusion of any transient increases in ODC activity due to surgery, animals were given ECS 10 days after adrenalectomy or sham operations. Data are mean k SE (bars) values from six to eight individual determinations. Values significantly different from basal levels using a two-tailed Student's t test after analysis of variance are indicated: ' p < 0.01. FollowingECS, there was no significant difference in ODC activity between adrenalectomized and sham-operated groups.
J. Neurochem., VoL 58. No. 2. 1992
CEREBELLUM
FIG. 4. Effect of MK-801 on ECS-induced ODC activity in various brain regions. Data are mean -t SE (bars) values from six to eight individualdeterminations. Values significantlydifferent from basal levels using a two-tailed Student's t test after analysis of variance are indicated: ' p < 0.01. MK-801 plus ECS values significantly different from the corresponding ECS only levels are also indicated: t p < 0.01.
T T
FIG. 2. Northern blots (top) and laser densitometricanalysis (bottom) of ODC mRNA in various brain regions 5 h following ECS.
NEOCORTEX
HIPPOCAMPUS
MK-801 +ECS
The induction of ODC activity in the neocortex is characterized by a proportionally enhanced level of transcription of the ODC mRNA (Zawia and Bondy, 1990). In contrast, induction of ODC activity in the hippocampus, which exhibits the highest basal value and is elevated the most by electrical stimulation, is independent of any enhanced transcription of ODC mRNA (Figs. 1 and 2). Even though part of the increase in ODC activity in the hippocampus may be attributed to this region's susceptibility to seizures, it is nonetheless apparent that the mode of induction of ODC activity is different here from that in the neocortex. Elevation of ODC activity in the cerebellum is also independent of any enhanced transcription of the ODC mRNA. However, it is not yet ascertained if the mechanism of ODC induction in the cerebellum is similar to that in the hippocampus. The primary stimuli and signals within the brain that lead to the elevation of ODC activity appear to be the same. The inability of adrenalectomy to alter the induction of ODC activity, following ECS, suggeststhat the induction of ODC activity in all brain regions is neuronally mediated. Furthermore, MK-80 1 uniformly depressed the induction of ODC activity. Hence, the ECS-initiated induction of ODC activity in all brain regions may be basically dependent on neuronal activity. However, the intermediate steps that lead to the elevation of ODC activity are clearly dissimilar in differing regions. These data suggest that ECS-induced depolarization can lead to the elevation of cerebral ODC activity in one of at least two ways: (a) In the neocortex, it can lead to genetic removal of inhibition of the ODC gene, leading to a rise in the levels of the ODC mRNA. The enhanced mRNA levels may then produce an elevation in ODC activity, through the de novo synthesis of new enzyme molecules. It is noteworthy that the induction of the ODC mRNA in the neocortex is a selective one, because the expression of the actin gene is not altered
DIFFERENTIAL REGULATION OF CEREBRAL ODC in response to ECS (Zawia and Bondy, 1990). (b) In the hippocampus and cerebellum, the same stimulus can lead to elevated ODC enzyme activity by acting on cytoplasmic targets. The mechanism in this case may involve changes in either the rate of synthesis or decay of the ODC protein. These mechanisms can only be confirmed if the levels of the ODC protein are measured using antibodies to the enzyme protein. Further investigations are underway to correlate the changes in ODC enzyme activity and mRNA to the levels of the ODC protein. It is not clear why there is a need for such alternate modes of ODC regulation in the brain. Such multiplicity of cerebral ODC regulation may be related to differential requirements of each brain region, concerning the speed and extent to which polyamine levels must be able to respond to altered environmental conditions. Acknowledgment: We would like to thank Dr. P. Coffino cDNA. This research was supported by grant ES0407 1 from the National Institutes of Health. for the ODC
REFERENCES Agnati L. F., Fuxe K., Zoli M., Davalli P., Corti A,, Zini I., and Toffano G. (1985) Effects of neurotoxic and mechanical lesions of the mesostriatal dopamine pathway on striatal polyamine levels in the rat: modulation by chronic ganglioside GM, treatment. Neurosci. Lett. 61, 339-344. Bondy S., Mitchell C., Rahmaan S., and Mason G. (1987) Regional variation in the response of cerebral ornithine decarboxylase to electroconvulsive shock. Neurochem. Pathol. 7, 129-141. Dam A. M. (1982) Hippocampal neuron loss in epilepsy and after experimental seizures. Acta Neurol. Scand. 66, 606-642. Dienel G. A. and Cruz N. F. (1984) Induction of ornithine decarboxylase during recovery from metabolic, mechanical, thermal, or chemical injury. J. Neurochem. 42, 1053-1061. Dornay M., Gilad V., Shiler I., and Gilad G. (1986) Early polyamine treatment accelerates regeneration of rat sympathetic neurons. Exp. Neurol. 92, 665-674. Fourney R., Miyakoshi J., Day R., and Paterson M. (1988) Northern blotting: efficient RNA staining and transfer. Focus 10, 5-7. Gilad G. and Gilad V. (1988) Early polyamine treatment enhances
739
survival of sympathetic neurons after postnatal axonal injury or immunosympathectomy. Dev. Brain Res. 38, 175- 18 1. Kanje M., Fransson I., Edstrom A., and Lowkvist B. (1986) Ornithine decarboxylaseactivity in dorsal root ganglia of regenerating frog sciatic nerve. Brain Res. 381, 24-28. Koenig H., Goldstone A., and Lu C. Y. (1983) Polyamines regulate calcium fluxes in a rapid plasma membrane response. Nature 305,530-534. Maniatis T., Fritsch E., and Sambrook J. (1982) Extraction, purification, and analysis ofmRNA from eukaryoticcells, in Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. McCann P. P. (1980) Regulation of ornithine decarboxylase in eukaryotes, in Polyamines in Biomedical Research (Gaugas J. M., ed), pp. 109-123. John Wiley & Sons, New York. McConlogue L., Gupta M., Wu L., and Coffino P. (1984) Molecular cloning and expression of the mouse ornithine decarboxylase gene. Proc. Natl. Acad. Sci. USA 81, 540-544. Metcalf B. W., Bey P., Danzin C., Jung M. J., Casara P., and Vevert J. P. (1978) Catalytic irreversible inhibition of mammalian ornithine decarboxylase by substrate and product analogues. J. Am. Chem. SOC.100,2551-2552. Pajunen A,, Hietala O., Virransalo E., and Piha R. (1978) Ornithine decarboxylaseand adenosylmethionine decarboxylasein mouse brain-effect of electrical stimulation. J. Neurochem. 30, 28 I 283. Russell D. H. and Snyder S. H. (1969)Amine synthesis in regenerating rat liver-extremely rapid turnover of ornithine decarboxylase. Proc. Natl. Acad. Sci. USA 60, 1420-1427. Slotkin T. A. and Bartolome J. (1986) Role of ornithine decarboxylase and the polyamines in nervous system development: a review. Brain Res. Bull. 17, 307-320. Soreq H., Safran A., and Eliyahu D. (1983) Modified composition of major ontogenetically regulated mRNAs and proteins in the cerebellum of old and of staggerer mice. Dev. Brain Res. 10, 73-82. Thomas P. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 17,5201-5205. Wells M. (1987) Changes of ornithine decarboxylase activity in dorsal root ganglion cells after axon injury: possible relationship to alterations in neuronal chromatin. Exp. Neurol. 95, 3 13-322. Wong E. H. F., Kemp J. A., Preistley T., Knight A. R., Woodruff G. N., and lversen L. L. (1986) The anticonvulsant MK-801 is a potent N-methyl-D-aspartateantagonist. Proc. Natl. Acad. Sci. USA 83, 7104-7108. Zawia N. H. and Bondy S. C. (1990) Activity-dependent rapid gene expression in the brain: ornithine decarboxylaseand c-fos. Mol. Brain Res. I, 243-247.
J. Neurochem , Vol. 58, NO. 2, 1992
Transcription-Dependent and -Independent Induction of Cerebral Ornithine Decarboxylase N.H. Zawia and S. C. Bondy Departments of Pharmacology and Community and Environmental Medicine and The Southern Occupational Health Center, University of CalSfornia,Irvine, California, U.S.A.
Abstract: Ornithine decarboxylase ( O X ; EC 4.I. 1.17) is a highly inducible, rate-limiting enzyme of the polyamine pathway. We have studied the mechanisms that lead to the induction of ODC activity in response to electrical stimulation in three brain regions. Hippocampal ODC activity was found to exhibit much larger elevations than that of the neocortex and the cerebellum. The levels of ODC gene expression were also followed to examine its relationship to the existing regional differences in ODC activity. In the neocortex, there was an elevation of both the ODC mRNA and enzyme activity. However, the hippocampal ODC mRNA level was not increased by electroconvulsive shock. Furthermore, the effects of hormonaI changes and seizures on these regional differ-
ences in ODC induction were also examined. Adrenalectomy did not affect ODC activity, but pretreatment with the anticonvulsant MK-801 caused a depression of the induced levels of enzyme activity. Our data suggest that ODC activity in all the brain regions studied is directly elevated by electrically stimulated seizures. However, this induced ODC activity may or may not involve enhanced gene expression. Key Words: Ornithine decarboxylase-mRNA-Electrical stimulation-Neocortex-Hippocampus-Induction. Zawia N. H. and Bondy S. C. Transcription-dependent and -independent induction of cerebral ornithine decarboxylase. J. Neurochem. 58, 736-739 (1992).
Induction of the enzyme ornithine decarboxylase (ODC; EC 4.1.1.17) is one of the earliest events that occur following perturbations to neural tissue (Koenig et al., 1983; Dornay et al., 1986; Wells, 1987; Gilad and Gilad, 1988). The elevation of ODC activity, following tissue damage, results in increased polyamine levels, which are believed to play a key role in the adaptive mechanisms of the CNS (Dienel and Cruz, 1984; Agnati et al., 1985; Kanje et al., 1986; Slotkin and Bartolome, 1986). The adult brain has a very low level of ODC activity basally, but this enzyme is highly inducible. Among the factors that play a significant role in the regulation of ODC activity are mRNA transcription and the turnover rate of the ODC protein (McCann, 1980). Induction of cerebral ODC activity by electrical stimulation has been reported (Pajunen et al., 1978). The hippocampus exhibits the greatest increases in ODC enzyme activity, in response to electroconvulsive shock (ECS), followed by the neocortex and the cerebellum (Bondy et al., 1987). However, it was not clear whether these varying degrees of ODC induction are
the outcome of the same process, or whether they reflect inherently different regional modes of ODC regulation. Recently we have shown that the induction of neocortical ODC activity by ECS is accompanied by a rapid elevation in the levels of the ODC mRNA (Zawia and Bondy, 1990). Here, we examine the regulation of ODC activity in response to electrical stimulation in three brain regions: neocortex, hippocampus, and cerebellum. At the molecular level, the rise in ODC activity has been compared with changes in the levels of the ODC mRNA. Furthermore, some factors potentially responsible for the fluctuations in ODC activity in each brain region have also been investigated. To this end, the modulation of the stimulated regional ODC activity by adrenalectomy or pretreatment with the anticonvulsant (+)-5-methyl-l0,1 l-dihydro-5H-dibenzo[u,d]cyclohepten-5,lO-imine (MK-80 1) were examined.
Received March 6 , 199I ; accepted July 17, I99 1. Address correspondence and reprint requests to Dr. N. H. Zawia at his present address: National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A.
Abbreviations used ECS, electroconvulsive shock; MK-801, (+)5-methyl-l0,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine; O K , ornithine decarboxylase.
MATERIALS AND METHODS Animals and treatment Male Fischer CD rats, 6-10 weeks old, were used. The animals were housed at 22°C with free access to food and
736
DIFFERENTIAL REGULATION OF CEREBRAL ODC water, on a 12-h light-dark cycle. ECS was administered via ear electrodes, with saline-moistened contact pads, using a constant-current apparatus. Shock was applied to normal, adrenalectomized (Zivic-Miller, Zelinople, PA, U.S.A.), and sham-operated (surgery performed but no adrenal glands removed) rats at the following intensity: 85 mA for 1 s with 1ms pulses at a frequency of 50 Hz. Control animals were treated similarly without the application of a current. These shock intensities have been demonstrated to be without detectable morphological damage to the brain (Dam, 1982). Some animals were dosed by intraperitoneal injections of the agent MK-80 1 (1 mg/kg), dissolved in saline. This agent is a selective antagonist of the N-methyl-D-aspartate-type of glutamate receptor (Wong et al., 1986). Animals were decapitated, and their corresponding brain regions were dissected out, quickly placed in dry ice, and kept at -70°C until use.
737
T
Neocortex
Hippocampus
Cerebellum
FIG. 1. Activities of ODC in different brain regions 5 h after ECS. Data are mean f SE (bars) values from seven to nine individual
determinations. Values significantly different from corresponding basal values using a two-tailed Student's t test are indicated: ' p < 0.001.
ODC assay ODC activity was determined by measurement of evolved I4CO2from carboxy [14C]ornithine(55.9 mCi/nmol; New England Nuclear, Boston, MA, U.S.A.). Tissue was homogenized in 19 volumes of 0.04 M Tns-HCI. After centrifugation (26,000 g,20 min), 0.9 ml of various tissue preparations was added to 50 pl of pyridoxal phosphate solution (1 nM) and 50 p1 of [I4C]ornithine, in the presence of 0.045 M dithiothreitol. The final ornithine concentration was 2.5 FM. Incubations were carried out at 37°C for 45 min in a sealed tube and terminated by injection of 1 ml of 2 M acetic acid into the reaction mixture (Russell and Snyder, 1969).Evolved I4CO2was trapped on a paper wick containing hyamine suspended above the reaction mixture. The decarboxylation process is linear for up to 1.5 h under these conditions. Decarboxylation not attributable to ODC was determined by assaying a parallel incubation in the presence of 5 W d i f l u oromethylornithine, a specific inhibitor of ODC (Metcalf et al., 1978).
RNA analysis Total cellular RNA was isolated from brain regions of control and ECS-treated animals using the phenol-sodium dodecyl sulfate method (Maniatis et al., 1982;Soreq et al., 1983). The RNA was fractionated according to size in formaldehydecontaining agarose gels (Fourney et al., 1988) and transferred onto nitrocellulose filter paper. The filters were then probed with a nick-translation 32P-labeled ODC cDNA plasrnid (McConlogue et al., 1984), washed under stringent conditions, and exposed to x-ray film at -70°C for 5-7 days (Thomas, 1980). The mRNA content was estimated by scanning the autoradiograms with a laser densitometer (LKB, Pharmacia), interfaced with a computer integrator. The areas under each peak were integrated to provide a quantitative estimate of the levels of the ODC mRNA.
RESULTS ODC activity Basal ODC activity in the hippocampus is twice as high as that in the neocortex and much higher than that in the cerebellum (Fig. I). ECS resulted in regional differences in the induction of ODC activity in the brain. The highest elevation in activity was seen in the hippocampus (fivefold), followed by the neocortex (twofold), whereas the cerebellum exhibited the least
stimulation of ODC activity (1.5-fold). These enzyme assays of ODC activity from different brain regions were consistent with earlier reports (Bondy et al., 1987).
ODC mRNA The basal ODC mRNA levels were similar in all brain regions examined (Fig. 2). ECS also produced regional differences in the induction of the ODC mRNA (Fig. 2). In contrast to the dramatic induction of enzyme activity in the hippocampus, the ODC mRNA levels did not change significantly in this brain region following ECS (Fig. 2). There was also no change in the level of the ODC message in the cerebellum. The neocortex, on the other hand, as previously shown, exhibited a significant elevation in the levels of ODC mRNA following ECS (Zawia and Bondy, 1990). Adrenalectomy and MK-801 Adrenalectomy had no significant effect on ECS-induced enzyme activity in any of the brain regions studied (Fig. 3). On the other hand, MK-80 1 pretreatment, which resulted in the abolition of the tonic-clonic seizures produced by ECS, effected a 40% attenuation of the ECS-induced ODC activity in all the areas surveyed (Fig. 4). Therefore, the induction of ODC was directly related to seizure activity.
DISCUSSION It is tempting to generalize that the regulation of an enzyme would be similar in all regions of the same organ. However, such an assumption is not true for cerebral ODC. The basal ODC activities exhibit some regional specificity, whereas the levels of the ODC mRNA appear to be similar in all brain regions examined (Figs. 1 and 2). Because the genomic capacity for ODC synthesis in all the brain regions appears to be the same, this suggests that some additional cytoplasmic components are involved in varying these regional levels of ODC activity, especially in maintaining the relatively high basal levels in the hippocampus. J. Neurochem.. Vol. 58. No. 2, 1992
N . H. ZAWIA A N D S. C. BONDY
738
T 0
BasalLevel ECS only
m
NEOCORTEX
0
BasalmRNA InducedmRNA
T T
NEOCORTEX
HIPPOCAMPUS
CEREBELLUM
Autoradiograms exhibiting ODC mRNA bands were scanned, and the intensity of the bands per integrated area was used to quantify the results. The autoradiograms shown are from two separate representative determinations.The autoradiograms with the basal (b) and ECS-induced (ecs) hippocampal ODC mRNA levels were exposed for a longer time to enhance the detection of the otherwise low ODC mRNA levels. In this blot, a lane of the basal neocortex (ntx) levels was included to show that the basal ODC mRNA levels in the two regions are similar. Data are mean It_ SE (bars) values from four to six individual determinations. Each determinationof hippocampal mRNA levels consisted of pooled tissue from several animals. Values significantly different from basal values using a two-tailed Student's t test are indicated: ' p < 0.05.
z "1
HIPPOCAMPUS
CEREBELLUM
FIG. 3. Effect of adrenalectomy on ECS-induced ODC activity in
various brain regions. To avoid the inclusion of any transient increases in ODC activity due to surgery, animals were given ECS 10 days after adrenalectomy or sham operations. Data are mean k SE (bars) values from six to eight individual determinations. Values significantly different from basal levels using a two-tailed Student's t test after analysis of variance are indicated: ' p < 0.01. FollowingECS, there was no significant difference in ODC activity between adrenalectomized and sham-operated groups.
J. Neurochem., VoL 58. No. 2. 1992
CEREBELLUM
FIG. 4. Effect of MK-801 on ECS-induced ODC activity in various brain regions. Data are mean -t SE (bars) values from six to eight individualdeterminations. Values significantlydifferent from basal levels using a two-tailed Student's t test after analysis of variance are indicated: ' p < 0.01. MK-801 plus ECS values significantly different from the corresponding ECS only levels are also indicated: t p < 0.01.
T T
FIG. 2. Northern blots (top) and laser densitometricanalysis (bottom) of ODC mRNA in various brain regions 5 h following ECS.
NEOCORTEX
HIPPOCAMPUS
MK-801 +ECS
The induction of ODC activity in the neocortex is characterized by a proportionally enhanced level of transcription of the ODC mRNA (Zawia and Bondy, 1990). In contrast, induction of ODC activity in the hippocampus, which exhibits the highest basal value and is elevated the most by electrical stimulation, is independent of any enhanced transcription of ODC mRNA (Figs. 1 and 2). Even though part of the increase in ODC activity in the hippocampus may be attributed to this region's susceptibility to seizures, it is nonetheless apparent that the mode of induction of ODC activity is different here from that in the neocortex. Elevation of ODC activity in the cerebellum is also independent of any enhanced transcription of the ODC mRNA. However, it is not yet ascertained if the mechanism of ODC induction in the cerebellum is similar to that in the hippocampus. The primary stimuli and signals within the brain that lead to the elevation of ODC activity appear to be the same. The inability of adrenalectomy to alter the induction of ODC activity, following ECS, suggeststhat the induction of ODC activity in all brain regions is neuronally mediated. Furthermore, MK-80 1 uniformly depressed the induction of ODC activity. Hence, the ECS-initiated induction of ODC activity in all brain regions may be basically dependent on neuronal activity. However, the intermediate steps that lead to the elevation of ODC activity are clearly dissimilar in differing regions. These data suggest that ECS-induced depolarization can lead to the elevation of cerebral ODC activity in one of at least two ways: (a) In the neocortex, it can lead to genetic removal of inhibition of the ODC gene, leading to a rise in the levels of the ODC mRNA. The enhanced mRNA levels may then produce an elevation in ODC activity, through the de novo synthesis of new enzyme molecules. It is noteworthy that the induction of the ODC mRNA in the neocortex is a selective one, because the expression of the actin gene is not altered
DIFFERENTIAL REGULATION OF CEREBRAL ODC in response to ECS (Zawia and Bondy, 1990). (b) In the hippocampus and cerebellum, the same stimulus can lead to elevated ODC enzyme activity by acting on cytoplasmic targets. The mechanism in this case may involve changes in either the rate of synthesis or decay of the ODC protein. These mechanisms can only be confirmed if the levels of the ODC protein are measured using antibodies to the enzyme protein. Further investigations are underway to correlate the changes in ODC enzyme activity and mRNA to the levels of the ODC protein. It is not clear why there is a need for such alternate modes of ODC regulation in the brain. Such multiplicity of cerebral ODC regulation may be related to differential requirements of each brain region, concerning the speed and extent to which polyamine levels must be able to respond to altered environmental conditions. Acknowledgment: We would like to thank Dr. P. Coffino cDNA. This research was supported by grant ES0407 1 from the National Institutes of Health. for the ODC
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J. Neurochem , Vol. 58, NO. 2, 1992
