By
From the * Laboratory of Biomedical Science, The Picower Institute for Medical Research, Manhasset,
New York 11030; the To elucidate endogenous mechanisms underlying cerebral damage during ischemia, brain
polyamine oxidase activity was measured in rats subjected to permanent occlusion of the middle cerebral artery. Brain polyamine oxidase activity was increased significantly within 2 h after
the onset of ischemia in brain homogenates (15.8 ± 0.9 nmol/h/mg protein) as compared with
homogenates prepared from the normally perfused contralateral side (7.4 ± 0.5 nmol/h/mg protein) (P <0.05). The major catabolic products of polyamine oxidase are putrescine and
3-aminopropanal. Although 3-aminopropanal is a potent cytotoxin, essential information was
previously lacking on whether 3-aminopropanal is produced during cerebral ischemia. We
now report that 3-aminopropanal accumulates in the ischemic brain within 2 h after permanent
forebrain ischemia in rats. Cytotoxic levels of 3-aminopropanal are achieved before the onset
of significant cerebral cell damage, and increase in a time-dependent manner with spreading
neuronal and glial cell death. Glial cell cultures exposed to 3-aminopropanal undergo apoptosis
(LD50 = 160 µM), whereas neurons are killed by necrotic mechanisms (LD50 = 90 µM). The
tetrapeptide caspase 1 inhibitor (Ac-YVAD-CMK) prevents 3-aminopropanal-mediated apoptosis in glial cells. Finally, treatment of rats with two structurally distinct inhibitors of
polyamine oxidase (aminoguanidine and chloroquine) attenuates brain polyamine oxidase activity, prevents the production of 3-aminopropanal, and significantly protects against the development of ischemic brain damage in vivo. Considered together, these results indicate that
polyamine oxidase-derived 3-aminopropanal is a mediator of the brain damaging sequelae of cerebral ischemia, which can be therapeutically modulated.
Cerebral ischemia, a leading cause of disability and mortality world-wide, is mediated by a cascade of molecular
cytotoxins that kill potentially viable cells in the brain. The
polyamines spermine, spermidine, and putrescine, which are
among the most abundant molecules in mammalian brain,
have been implicated in the pathogenesis of ischemic brain
damage (1). Polyamine biosynthesis is increased after the
onset of cerebral ischemia, due to an ischemia-mediated induction of ornithine decarboxylase, a key synthetic enzyme
in the polyamine biosynthetic pathway (9). Spermine
was recently linked to the development of glutamate-mediated cytotoxicity, because it can bind to the NR1 subunit
of the NMDA receptor and potentiate glutamate-mediated
cell damage (14). Administration of experimental therapeutics that inhibit ornithine decarboxylase prevents the
development of ischemic brain damage, suggesting that the
accumulation of polyamines in the ischemic brain occupies an important role in the pathogenesis of stroke (9).
Somewhat paradoxically, however, brain spermine and
spermidine levels decrease during cerebral ischemia (5, 18,
19). This decline of tissue spermine and spermidine levels is
accompanied by an increase in brain putrescine levels (13,
19). Furthermore, intracerebral putrescine levels correlate significantly with the volume of dead brain, suggesting
that putrescine may be an endogenous molecular marker
for the extent of ischemia-induced damage. Notably, putrescine does not interact with the N-methyl-D-aspartate
(NMDA) receptor, and does not potentiate its function.
Therefore, we reasoned that a possible explanation for
these results could be found in the catabolism of polyamines
via the "interconversion pathway", which is dependent
upon the activity of tissue polyamine oxidase (20, 22).
This ubiquitous enzyme, which is present in high levels in
brain and other mammalian tissues, cleaves spermine and
spermidine via oxidative deamination to generate the end
products putrescine and 3-aminopropanal (22, 26).
3-Aminopropanal is widely known for its cytotoxicity to
primary endothelial cells, fibroblasts, and a variety of transformed mammalian cell lines (29). 3-Aminopropanal has
also been implicated as a mediator of programmed cell death
in murine embryonic limb buds, and may contribute to the
development of necrosis in some tumors (34, 35). Inhibition
of polyamine oxidase with aminoguanidine, a well-characterized inhibitor, blocks the generation of 3-aminopropanal in cell cultures after the addition of spermine, and prevents subsequent cytotoxicity (30, 36, 37). The LD50 concentration of 3-aminopropanal to cells is similar to that of
glutamate excitotoxicity to neurons (38). In contrast, putrescine is not cytotoxic to cells (even in the millimolar
range) but its rate of production through polyamine oxidation correlates with the rate of formation of the cytotoxin
(3-aminopropanal). Reasoning that increased production of
3-aminopropanal might therefore mediate cytotoxic brain
damage during cerebral ischemia, we investigated the activity
of polyamine oxidase, and measured the levels of 3-aminopropanal in an animal model of cerebral infarction.
We now report that cerebral ischemia mediates the induction of brain polyamine oxidase activity, and that the cytotoxic
end product 3-aminopropanal accumulates in the brain at levels that are lethal to neurons and glial cells. In glial cells, 3-aminopropanal mediates apoptosis by activation of a caspase
1-dependent signaling pathway, whereas in neurons it causes
necrotic cell death. Inhibition of polyamine oxidase activity
with structurally distinct compounds prevents the formation
of 3-aminopropanal and provides significant protection
against the development of cerebral damage after permanent cerebral artery occlusion in rats. This is the first direct
evidence that polyamine oxidase-derived 3-aminopropanal
is a potential therapeutic target in cerebral ischemia.
Animal Model of Permanent Middle Cerebral Artery Occlusion.
Department of Physiology and the § Department of Neurology, University of
Minnesota, Minneapolis, Minnesota 55455; the
Department of Pediatrics, Baylor College of Medicine,
Houston, Texas 77030; the ¶ Department of Surgery, North Shore University Hospital-New York
University Medical School, Manhasset, New York 11030; the ** Department of Experimental
Pathology and Laboratory Medicine, Albany Medical College, Albany, New York 12208; and the
Division of Biology and Human Genetics,
Laboratory of Organic Chemistry, The Picower Institute for
Medical Research, Manhasset, New York 11030
Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Polyamine Oxidase Assay.
Polyamine oxidase in brain homogenate was assayed as previously described (25, 26, 42). In brief, 2 h after occlusion of the middle cerebral artery, a 4-mm-thick coronal section of ipsilateral hemisphere encompassing the zone of ischemia (beginning 3 mm caudal from the frontal pole) was manually homogenized on ice in 1.5 ml of Hanks media containing 1 mM PMSF, and was centrifuged at 43,000 g for 30 min. Brain polyamine oxidase activity in the homogenates was determined by addition of spermine to the homogenate at time zero (50 µl of a 1 mM stock solution added per 1 ml of supernatant). Where indicated in some experiments the enzyme inhibitors aminoguanidine or chloroquine (50 µM-5 mM) were added 5 min before spermine. Homogenates were maintained at 37°C, and duplicate 200-µl samples were removed at time points up to 60 min after the addition of spermine; enzyme activity in the samples was stopped by addition of 10 µl of 60% perchloric acid (PCA). Samples for HPLC analysis to detect spermine were prepared as described below. Enzyme activity was corrected for the protein content of the supernatants using a commercially available protein assay (Bio-Rad Protein Assay; Bio-Rad, Hercules, CA) with BSA (GIBCO BRL, Gaithersburg, MD) as a standard.3-Aminopropanal.
3-Aminopropanal was prepared by hydrolysis of 3-aminopropanal diethyl acetal (145 mM; TCI America, Portland, OR) in 1.5 M of HCl for 5 h at room temperature. The reaction mixture was applied to a column (3 × 6 cm) of Dowex-50 (H+ form; Sigma Chemical Co., St. Louis, MO) ion exchange resin and eluted with a step gradient of HCl (0-3 M; 160 ml; flow rate 0.7 ml/min). Fractions containing aldehyde, as determined by the method of Bachrach and Reches (43), were concentrated in a centrifugal evaporator at room temperature. The concentration of 3-aminopropanal was determined spectrophotometrically atDerivatization of 3-Aminopropanal with 2,4-Dinitrophenylhydrazine.
2,4-dinitrophenylhydrazine (0.5 g) in concentrated HCl/ ethanol (1:10, vol/vol; 11 ml) was refluxed for 10 s with the aqueous 3-aminopropanal. The resulting 3-aminopropionaldehyde- 2,4-dinitrophenylhydrazone derivative was precipitated at room temperature and collected by filtration. 1H-nuclear magnetic resonance (NMR) spectroscopy (DMSO-d6 and CDCl3, 270 MHz) of purified 2,4-dinitrophenylhydrazone derivative was employed to confirm its structure. The NMR spectrum revealed the presence of syn and anti isomers (1:1) with resonance atHPLC Detection of the Derivatization Products of 3-Aminopropanal and 2,4-Dinitrophenylhydrazine.
A liquid chromatograph (model 1090; Hewlett-Packard, Wilmington, DE), equipped with an autosampler, photo diode-array, fluorescence detectors, and Chemstation operating software, was used for all analyses. We used detection by fluorescence, based on the reaction of 5-dimethyl-aminonapthalene sulfonyl-chloride (dansyl chloride; Molecular Probes, Eugene, OR; relative fluorescence intensity 280-340 out of 430 nm) with primary and secondary amines. Dansylation was performed by reacting 50 µl of the sample with 200 µl of 10 mg/ ml dansyl chloride solution in acetone, 200 µl of saturated Na2CO3 solution, 3 µl of 60% PCA, and 3 µl of 1-mM 1,7-diaminoheptane (Sigma Chemical Co.), followed by incubation at 65°C for 10 min. 20 µl of the resulting supernatant was injected onto a C-4 250 × 4.6 mm column (Vydac, Hesperia, CA) with 5-µm particle size. Using a flow rate of 1.0 ml/min, runs were initiated at 100% A (dH2O) and a linear gradient to 100% B (methanol) performed over 45 min, followed by 5 min of 100% B and a return to 100% A over 5 min. For detection of the presence of 3-aminopropanal in ischemic brain, animals were subjected to permanent middle cerebral artery occlusion and killed at the times indicated. Brain sections corresponding to the area of focal infarction caused by middle cerebral artery occlusion (4-mm-thick located 3 mm caudal to the frontal lobes) were quickly excised. Control brain slices were taken from sham-operated animals. Manual homogenization was performed in 1.5 ml of 2,4-dinitrophenylhydrazine reagent followed by concentration and HPLC analysis as described above. The limit of detection of 3-aminopropanal with this assay is 180- 200 nmol/ml. Results are normalized for protein content using a commercially available assay (Bio-Rad Protein Assay; Bio-Rad) and corrected for HPLC injection volume using an internal standard of 1,7-diaminoheptane.Stereotactically Guided Microinjections of Polyamines into the Cerebral Cortex.
Male Lewis rats (270-300 g) were anesthetized and placed in a stereotactic head frame (Stoelting Co., Wood Dale, IL). The incisor bar was adjusted until the plane defined by the lambda and bregma was parallel to the base plate. A microsurgical craniotomy was performed 1.7 mm anterior to bregma, and 5 mm right of the midline, and the tip of a 29-gauge needle was advanced 2 mm deep to the dural opening. Polyamine- or 3-aminopropanal-containing solutions (25 µg) prepared in 2 µl of sterile saline (NaCl; 154 mM) were injected over 3 min, and the needle was left undisturbed for 5 min and then removed. Animals were killed 48 h later, and the brains were excised and sectioned in 1-mm-thick slices in the coronal plane, and then immersed for 30 min at 37°C in a solution containing 2,3,5-triphenyl-2H-tetrazolium chloride (2% in NaCl; 154 mM). Brain infarction was visualized as areas of unstained (white) tissue which were easily contrasted with viable tissue (stained red). Slices were placed in buffered 10% formalin and infarct size was quantitatively assessed by planimetric analysis. In separate studies, histopathological analysis of brain sections verified the location of the injectate and the correlation of necrosis revealed by TTC staining. Groups of three or four animals were used for each of the experimental conditions as noted.Tissue Culture.
The glial (HTB14; reference 45) and neuronal (HTB11; reference 46) cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in DMEM (GIBCO BRL) containing fetal bovine serum (10%; Hyclone, Logan, UT), sodium pyruvate (1 mM; Sigma Chemical Co.), penicillin and streptomycin (0.5%; Sigma Chemical Co.) in a humidified atmosphere (5% CO2; 37°C). For all experiments involving exposure to 3-aminopropanal, cells were grown in 96-well microtiter plates to 90-95% confluence and medium was replaced with fresh serum-free medium (Opti-MEM I; GIBCO BRL) to prevent nonspecific interaction of 3-aminopropanal with serum proteins. For all experiments using a short duration of 3-aminopropanal exposure (5 min to 2 h in 96-well plates), the cells were washed at the times indicated, and then incubated in Opti-MEM I for up to 20 h. Where indicated, cells were pretreated with the caspase 1 inhibitor II Ac-YVAD-CMK (BACHEM, Torrance, CA) or the caspase-3 inhibitor Ac-DEVD-CHO (Peptides International, Louisville, KY) in DMSO for 3 h followed by addition of 3-aminopropanal for an additional 5 h. DMSO vehicle controls were performed concurrently. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Chemical Co.) as previously described (47). Data are expressed as mean ± SE; n = 3-6 wells per condition; experiments were performed in triplicate.TUNEL Staining by FACS®.
Cells were treated with 3-aminopropanal as indicated and then harvested by centrifugation (1,500 rpm for 5 min). The pellets were fixed with 1× ORTHO Permeafix (Orthodiagnostics, Raritan, NJ) at room temperature for 40 min. After washing with Dulbecco's PBS containing 1% BSA (PBS-BSA), cells were stained by the TUNEL (Tdt-mediated dUTP-biotin nick-end labeling) method using the ApopTag Direct Fluorescein kit (Oncor, Gaithersburg, MD). Negative controls were performed using a reaction mixture devoid of TdT. A FACScan® (Becton Dickinson, Sunnyvale, CA) was used for all analyses; 5,000-10,000 events (ungated) were collected using single color histogram for FITC.Annexin V/Propidium Iodide Staining.
Annexin V/propidium iodide (PI) staining was performed using a commercially available kit (The Apoptosis Detection Kit; R&D Systems, Minneapolis, MN). Cells were analyzed by flow cytometry within 1 h of completion of staining.DNA Electrophoresis.
HTB11 or HTB14 cells were harvested (2-3 × 107 cells) by centrifugation (1,000 rpm for 5 min), resuspended in a reaction buffer containing proteinase K, and incubated overnight at 55°C. RNAase was added to a final concentration of 50 µg/ml, and samples were incubated at 37°C for 1 h. DNA was extracted three times with phenol/chloroform and two times with chloroform and precipitated in 2 vol of 100% cold ethanol and 0.3 M of sodium acetate (pH 5.2). DNA was resuspended in 50 µl of dH2O, fractionated by 1.5% agarose gel electrophoresis, and stained with SYBR Green I nucleic acid stain (Molecular Probes). ![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To determine the role of brain polyamine oxidase activity in cerebral ischemia, Lewis rats were subjected to focal cerebral infarction by microsurgical occlusion of the middle cerebral artery in a standardized model as described previously (39, 40, 48). Brain homogenates were prepared from the anatomic region perfused by the middle cerebral artery, and total polyamine oxidase (PAO) activity was determined using a method described previously (25, 26, 42). Polyamine oxidase activity was significantly higher in homogenates prepared from ischemic hemispheres as compared with normally perfused contralateral hemispheres (PAO activity after ischemia = 15.8 ± 0.9 nmol/h/mg protein; versus PAO activity in sham-operated controls = 7.4 ± 0.5 nmol/h/mg protein; P <0.05; Fig. 1). The increase of brain polyamine oxidase activity was detected within 2 h after the onset of cerebral ischemia. Two structurally distinct inhibitors of polyamine oxidase activity (aminoguanidine and chloroquine) were used to assess specificity (26, 49). Addition of either agent to the ischemic brain homogenates dose-dependently inhibited polyamine oxidase activity; chloroquine, IC50 = 40 µM; aminoguanidine, IC50 = 400 µM. This indicates that within 2 h after the onset of cerebral ischemia there is a specific induction of brain polyamine oxidase activity, and that this activity can be pharmacologically inhibited.
|
To obtain direct evidence that the cytotoxin 3-aminopropanal is produced during cerebral ischemia, we developed a method to detect brain 3-aminopropanal using HPLC and mass spectroscopy. 3-Aminopropanal was prepared by hydrolysis of the diethyl acetal and then derivatized using 2,4-dinitrophenylhydrazine (Fig. 2 a). HPLC analysis of the derivatized products revealed two peaks (at 24 and 27 min, respectively) in equal ratio (Fig. 2 b, inset). H-NMR spectroscopy revealed the presence of anti and syn isomers, as predicted by the structures of the principle condensation products (Fig. 2 a). Electrospray ionization mass spectroscopy (EIMS) of the HPLC-purified products detected the expected mass ion m/z 251 (Fig. 2 b). We then subjected rats to permanent focal cerebral ischemia, and derivatized brain homogenates with 2,4-dinitrophenylhydrazine. HPLC analysis of the derivatized brain homogenate revealed the appearance of the two expected peaks, and EIMS confirmed identity as the isomeric 3-aminopropanal-2,4-dinitrophenylhydrazone reaction products (Fig. 2 c). The 3-aminopropanal derivatization products could not be detected in brain homogenates prepared from sham-operated, normally perfused control animals (Fig. 3). 3-Aminopropanal became significantly elevated within 2 h after the onset of ischemia, and increased in a time-dependent manner for at least 25 h after the onset of ischemia (Fig. 3). The HPLC assay we used may well have underestimated the amount of 3-aminopropanal produced in the ischemic brain, because 3-aminopropanal is a reactive molecule that can bind to the amino and sulfhydryl groups of proteins (33, 52), thereby decreasing its availability for derivatization and detection. Nonetheless, after correcting the measured levels for total brain protein (213 g/liter), brain 3-aminopropanal concentrations after ischemia reach a highly cytotoxic range (0.5- 2.7 mM). When considered with our previous observation that cerebral ischemia mediates an early induction in the activity of brain polyamine oxidase, these findings indicate that this enzyme pathway continually generates 3-aminopropanal during the first 25 h after the onset of cerebral ischemia.
|
|
We next examined whether the enzymatic formation of 3-aminopropanal preceded the onset of ischemic cell death. Accordingly, the volume of dead brain was measured by staining brain sections with the vital dye TTC. For the first 3 h of ischemia, cells in the region of the occluded middle cerebral artery were observed to be largely viable (total volume of cell death = 2 ± 2 mm3). Histological examination of hematoxylin and eosin- stained brain sections (data not shown) confirmed that cells were morphologically intact and had not yet developed degenerative changes at a time when 3-aminopropanal levels were already significantly increased (Fig. 3). Over the next 25 h, spreading cell death developed in association with increasing 3-aminopropanal levels (infarct volume at 25 h = 71 ± 24 mm3; versus infarct volume at 3 h = 2 ± 2 mm3; P <0.05). These findings give evidence that 3-aminopropanal accumulates during the early response to cerebral ischemia, and precedes the development of progressive, spreading brain cell death.
Brain Damage Is Mediated by Intracortical Microinjection of Spermine, Spermidine, or 3-Aminopropanal, but Not by Putrescine.Since polyamine oxidase activity is present in normal mammalian brain (Fig. 1 and references 22, 25), we wished to investigate whether increased extracellular levels of substrate (e.g., spermine or spermidine) would induce local cell death. Accordingly, spermine and spermidine were administered into rat cerebral cortex by direct stereotactic microinjection, and the volume of cell death was measured by TTC staining brain sections. We observed significant cortical cell death after spermine or spermidine administration, but not after that of putrescine, a polyamine that cannot be degraded by polyamine oxidase (Fig. 4 a). Microinjection of 3-aminopropanal mediated significant cell death in the cerebral cortex (Fig. 4 a). The quantity of 3-aminopropanal administered (25 µg/injection) is similar to the amounts endogenously produced during ischemia (~350 µM assuming a volume of distribution of a typical middle cerebral artery infarction in this model). Systemic administration of the polyamine oxidase inhibitors (chloroquine or aminoguanidine) conferred significant protection against the development of spermine-mediated intracortical damage (Fig. 4 b), suggesting that polyamine oxidase activity is necessary to mediate the cytotoxicity of extracellular spermine. Intracortical administration of aminoguanidine also conferred significant protection against intracortical spermine-mediated cell death (Fig. 4 b), indicating that the cerebroprotective effects of enzyme inhibition occur locally in brain, and not via some unanticipated peripheral drug action. Of note, aminoguanidine failed to significantly attenuate the direct cytotoxicity of intracortical 3-aminopropanal (Fig. 4 b), suggesting that the protective mechanism of aminoguanidine against spermine cytotoxicity is through inhibition of polyamine oxidase activity, and not through direct inhibition of 3-aminopropanal. Thus, increased extracellular levels of spermine, spermidine, or 3-aminopropanal (but not putrescine) are cytotoxic to cerebral cortical cells in vivo.
|
We performed histopathologic examination of brain sections 24 h after intracortical 3-aminopropanal microinjection and observed localized degenerative changes surrounding the injection zone (Fig. 5 A). Cells within the 3-aminopropanal injection site were necrotic, as evidenced by eosin-positive staining. Moreover, in the same region we also observed cells undergoing programmed cell death, as evidenced by TUNEL-positive staining (Fig. 5, B and C). These changes were not observed in the injection zone when either vehicle or putrescine was administered (data not shown). Direct intracortical administration of spermine also caused cell necrosis and apoptosis, and these findings were significantly inhibited by administration of aminoguanidine (data not shown). Thus, the accumulation of extracellular 3-aminopropanal induces brain cell damage in vivo, which occurs through both necrosis and programmed cell death.
|
To investigate directly the cytotoxic signaling mechanisms of 3-aminopropanal, we exposed cultured human glial (HTB14) and neuronal (HTB11) cell lines to 3-aminopropanal. After 20 h of incubation, the LD50's for 3-aminopropanal were 160 ± 10 µM for the glial cell line and 90 ± 20 µM for the neuronal cell line (HTB11). 3-Aminopropanal was somewhat more cytotoxic in primary rat astroglial cell cultures (LD50 = 80 ± 9 µM). A time-course study revealed that 3-aminopropanal exposure for as little as 5 min was significantly cytotoxic to neuronal cells, but a longer exposure was required to mediate significant cytotoxicity in glial cells (Table 1). This delayed onset suggested that glial cell death might be dependent upon apoptosis-mediated pathways, and in agreement with this possibility, we observed apoptosis-specific DNA fragmentation after exposure of glial cells to 3-aminopropanal (Fig. 6 a). We obtained additional evidence of apoptosis by flow cytometric detection of DNA strand breaks using the TUNEL method (53). In these experiments, 76% of the glial cells stained TUNEL-positive after 13 h of exposure to 160 µM of 3-aminopropanal (Fig. 6 b), whereas vehicle-treated control cells were uniformly negative (Fig. 6 c). Multiparameter flow cytometry revealed that glial cells exposed to 3-aminopropanal exhibited a decrease in cellular forward light scatter and an increase in side scatter, in agreement with typical cell shrinkage, chromatin condensation, and nuclear fragmentation. Apoptosis of glial cells was also confirmed by subdiploid staining with propidium iodide and annexin V/PI (data not shown).
|
|
In contrast to the results in the glial cell line, 3-aminopropanal did not induce apoptosis in neuronal cell cultures (HTB11) using similar experimental methods. DNA electrophoresis of 3-aminopropanal-treated neurons revealed no evidence of chromosomal DNA degradation (Fig. 6 a). In addition, we observed no increase in TUNEL positivity under these conditions, although a forward/side scatter analysis revealed significant cell death after 3-aminopropanal treatment (55.7%), but not in vehicle-treated controls (9.1%). There was also no evidence of apoptosis as measured with annexin V, a method used to detect loss of cell membrane phospholipid asymmetry that can be associated with apoptosis (Fig. 6, f and g). Apoptosis could be induced in neuronal cells by exposure to camptothecin (15 µg/ml for 20 h; reference 54) as assessed by TUNEL and Annexin V methods (data not shown), indicating that the absence of apoptosis after 3-aminopropanal exposure was not due to some unanticipated generalized cellular defect in this neuronal cell line. Thus, in contrast to glial cells, exposure of neurons to 3-aminopropanal causes primarily necrotic cell death.
Inhibition of Caspase 1 Prevents 3-Aminopropanal-induced Apoptosis in Glial Cells.The cysteine proteases caspase 1 and caspase 3 have been implicated in the cellular signaling pathways mediating apoptosis during cerebral ischemia (55- 59). To investigate whether these proteases were required for the induction of apoptosis by 3-aminopropanal in glial cells, HTB14 cells were treated for 3 h with a tetrapeptide caspase 1 inhibitor (Ac-YVAD-CMK), or with a caspase 3 inhibitor (Ac-DEVD-CHO), followed by a 5-h treatment with 3-aminopropanal. Treatment with the caspase 1 inhibitor, but not the caspase 3 inhibitor, conferred dose-dependent inhibition of 3-aminopropanal-induced cell death (Fig. 7, a and b). These data give evidence for a specific role of the caspase 1 proteases in the 3-aminopropanal-induced signaling that mediates apoptosis in glial cells.
|
The mechanism of ischemic brain cell damage proposed here predicts that administration of polyamine oxidase inhibitors during cerebral ischemia in vivo will reduce both the accumulation of 3-aminopropanal and the volume of cerebral infarction. Accordingly, we measured these end points after administering two structurally distinct polyamine oxidase inhibitors to rats in the standardized model of permanent middle cerebral artery occlusion. We reported previously that aminoguanidine administered after the onset of cerebral ischemia (320 mg/kg intraperitoneally 15 min after ischemia) significantly reduces the volume of cerebral damage (40). In this study we administered aminoguanidine by this established treatment protocol and observed that it effectively prevented the increase of brain 3-aminopropanal levels (Table 2). In agreement with the proposed mechanism of inhibiting polyamine oxidase, the administration of the structurally distinct enzyme inhibitor chloroquine also conferred effective cerebroprotection against ischemic cell damage, even when the administration was delayed 15 min after occlusion of the middle cerebral artery (Table 2). We previously reported that the protective effects of aminoguanidine are not attributable to altering peripheral cardiovascular parameters that influence the volume of brain damage (40). In this study, physiological parameters determined before and during ischemia (blood pressure, heart rate, body temperature, and arterial blood gases) did not differ among groups treated with vehicle or chloroquine (data not shown). Thus, the cerebroprotective effects of chloroquine cannot be attributed to altering the peripheral cardiovascular response to cerebral ischemia.
|
Previously, Zhang et al. reported that iNOS is upregulated 24-48 h after cerebral ischemia, and that delayed administration of aminoguanidine can prevent secondary NO-mediated brain damage in a delayed therapeutic window (60). Our results here indicate that polyamine oxidase activity is upregulated much earlier after cerebral ischemia (within 2 h), and that early administration of aminoguanidine inhibits the generation of 3-aminopropanal. Although the most direct interpretation of our results is that two structurally distinct inhibitors of polyamine oxidase prevent ischemic damage by preventing the formation of 3-aminopropanal, we nonetheless performed a series of additional experiments to exclude other possibilities.
First, we wished to exclude the unlikely possibility that chloroquine protection occurred through an unanticipated inhibition of iNOS. Addition of even suprapharmacological amounts of chloroquine (1 mM) failed to inhibit iNOS activity measured in murine macrophage-like RAW264.7 cell lysates (control iNOS activity = 13,254 ± 250 DPM/ µg protein; versus chloroquine iNOS activity = 11,755 ± 883 DPM/µg protein; P >0.05). We also wished to exclude the unlikely possibility that aminoguanidine or chloroquine might protect cells by directly inactivating the cytotoxicity of 3-aminopropanal. Cell cytotoxicity was measured in the presence of inhibitors, and we observed that the LD50 for 3-aminopropanal after overnight incubation in HTB11 cells was similar whether or not aminoguanidine or chloroquine were added (data not shown). We also wished to exclude the unlikely possibility that the mechanism of aminoguanidine protection is not mediated via altering the sensitivity of cells to the cytotoxicity of glutamate. When aminoguanidine was added to primary neuronal cultures treated with NMDA we observed no significant attenuation of cytotoxicity (Table 3). We also addressed whether 3-aminopropanal mediates cell death through induction of iNOS activity. Addition of iNOS inhibitors (L-NMMA or aminoguanidine) to 3-aminopropanal-treated glial cells failed to attenuate the development of TUNEL positivity as measured by FACS® (data not shown).
|
Although we have excluded a number of plausible alternative mechanisms through which aminoguanidine might protect against cerebral ischemia, it remains theoretically possible that other nonspecific activities of chloroquine might additionally contribute to the observed protection against infarction (i.e., inhibition of free radical formation, phospholipase activity, or protein synthesis). However, these mechanisms are not supported by our direct observations that (a) inhibiting polyamine oxidase activity reduces the formation of cytotoxic concentrations of 3-aminopropanal; (b) 3-aminopropanal cytotoxicity cannot be blocked with aminoguanidine or chloroquine; and (c) either chloroquine or aminoguanidine prevent the brain damaging effects of either intracortical spermine or ischemia.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Four closely related lines of evidence therefore support the role of 3-aminopropanal as a cytotoxic mediator of brain damage in cerebral ischemia. First, cerebral ischemia mediates an early induction of polyamine oxidase activity. Second, the cytotoxic enzyme product 3-aminopropanal accumulates during the early response to cerebral ischemia, but is not produced in normally perfused controls. Third, 3-aminopropanal production in the ischemic brain increases before the onset of significant cellular degeneration, with tissue 3-aminopropanal levels rising further during the period of progressive cell death. Fourth, 3-aminopropanal is a potent cytotoxin that activates apoptosis via a caspase 1-dependent mechanism in glial cells and necrosis in neurons. Considered together, these data offer an explanation for the correlation between brain levels of putrescine, a stable end-product of terminal polyamine oxidation, and infarct volume (5, 18, 61), since catabolism of spermine and spermidine by polyamine oxidase produces both a stable, nontoxic end product (putrescine) and a potent cytotoxin (3-aminopropanal). The latter product mediates cell death, and the former accumulates in correlation to the extent of damage.
Previous observations suggest that polyamines can prevent apoptosis in neuronal cultures (62, 63), or can amplify glutamate-mediated cell cytotoxicity (14). Cell survival in the ischemic zone is likely to be critically dependent upon the balance between the direct effects of polyamines and the cytotoxic effects of 3-aminopropanal. We have yet to address the contribution to brain damage of an alternative pathway of polyamine catabolism, the acetylation pathway, but it is reasonable to speculate that this pathway could provide an additional source of potentially toxic aldehyde products, e.g., 3-acetamidopropanal (64, 65). There has been some controversy as to whether both 3-aminopropanal and 3-acetamidopropanal can produce acrolein in vivo, a known mediator of cytotoxicity and apoptosis (66, 67). It is likely that several products of polyamine oxidation further augment the cytotoxicity of 3-aminopropanal. When considered together, these observations add credence to the previously uninvestigated hypothesis that enhanced polyamine oxidation during cerebral ischemia is deleterious.
Our results now suggest the following mechanism of
brain cell death during cerebral ischemia: dead and dying
cells in the densely h
ypoxic core release stores of intracellular spermine and
spermidine, which are catabolized by polyamine oxidase.
The resultant production of 3-aminopropanal causes apoptosis in surrounding glial cells, and necrosis of neurons,
which in turn release more spermine and spermidine as
substrate for polyamine oxidase. This cytotoxic mechanism
spreads to involve a larger volume of potentially viable cells
surrounding the ischemic core. It is likely that 3-aminopropanal is positioned proximally in the mediator cascade elicited by cerebral ischemia, which includes the excitatory
amino acids, activated oxygen species, nitric oxide, TNF,
IL-1, IL-6, and platelet-activating factor (48, 60, 68). It
is interesting to reconsider previous observations that expression of dominant negative mutants of IL-1 converting
enzyme (caspase 1) protects against the development of apoptosis during cerebral ischemia (58, 76). Based on our results, it is now plausible that inhibition of caspase 1 activity
prevents the damaging effects of 3-aminopropanal. Further,
we previously reported that TNF synthesis is upregulated during the first 12 h of brain ischemia, and that TNF participates in the mediation of brain damage (48). It will now
be interesting to explore further the influence of decreasing
brain spermine levels after ischemia, because spermine is a
direct inhibitor of TNF synthesis in human peripheral
blood mononuclear cells (77). We have recently found that
centrally administered 3-aminopropanal directly stimulates
intracerebral TNF synthesis (data not shown), indicating
that 3-aminopropanal may stimulate this component of the
ischemic cytotoxic cascade.
Polyamines and polyamine oxidase are ubiquitous in mammalian tissues (78). This poses the intriguing possibility that the mechanism of polyamine oxidative tissue damage proposed here might be invoked in other ischemic conditions, like myocardial infarction and tumor necrosis.
![]() |
Footnotes |
---|
Address correspondence to Kevin J. Tracey, The Picower Institute for Medical Research, 350 Community Dr., Manhasset, NY 11030. Phone: 516-562-9476; Fax: 516-562-2356; E-mail: ktracey{at}picower.edu
Received for publication 10 February 1998 and in revised form 6 May 1998.
Abbreviations used in this paper EIMS, Electrospray ionization mass spectroscopy; iNOS, inducible nitric oxide synthase; NMDA, N-methyl-D-aspartate; NMR, nuclear magnetic resonance; PAO, polyamine oxidase; PCA, perchloric acid; TTC, 2,3,5-triphenyltetrazolium chloride; TUNEL, Tdt-mediated dUTP-biotin nick-end labeling.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Zhang, L.,
X. Xheng,
M.C. Paupard,
A.P. Wang,
L. Santchi,
L.K. Friedman,
R.S. Zukin, and
M.V.L. Bennett.
1994.
Spermine potentiation of recombinant N-methyl-D-aspartate
receptors is affected by subunit composition.
Proc. Natl. Acad.
Sci. USA.
91:
10883-10887
|
2. | Harman, R.J., and G.G. Shaw. 1981. The spontaneous and evoked release of spermine from rat brain in vitro. Br. J. Pharmacol. 73: 165-174 [Abstract]. |
3. | Bergeron, R.J., W.R. Weimar, Q. Wu, Y. Feng, and J.S. McManis. 1996. Polyamine analogue regulation of NMDA MK-801 binding: a structure-activity study. J. Med. Chem. 39: 5257-5266 [Medline]. |
4. | Glantz, L., J.L. Nates, V. Trembovler, R. Bass, and E. Shohami. 1996. Polyamines induce blood-brain barrier disruption and edema formation in the rat. J. Basic Clin. Physiol. Pharmacol. 7: 1-10 [Medline]. |
5. | Dempsey, R.J., M.W. Roy, K. Meyer, H.H. Tain, and J.W. Olson. 1985. Polyamine and prostaglandin markers in focal cerebral ischemia. Neurosurgery (Baltim.). 17: 635-640 [Medline]. |
6. | Schmitz, M.P., D.J. Combs, and R.J. Dempsey. 1993. Difluoromethylornithine decreases postischemic brain edema and blood-brain barrier breakdown. Neurosurgery (Baltim.). 33: 882-888 [Medline]. |
7. | Anderson, D.J., J. Crossland, and G.G. Shaw. 1975. The actions of spermidine and spermine on the central nervous system. Neuropharmacology. 14: 571-577 [Medline]. |
8. | Doyle, K.M., and G.G. Shaw. 1994. The mechanism of the neurotoxic effects of spermidine. Biochem. Soc. Trans. 22 (Suppl.): 386S [Medline]. |
9. | Kindy, M.S., Y. Hu, and R.J. Dempsey. 1994. Blockade of ornithine decarboxylase enzyme protects against ischemic brain damage. J. Cereb. Blood Flow Metab. 14: 1040-1045 [Medline]. |
10. | Marton, L.J., and A.E. Pegg. 1995. Polyamines as targets for therapeutic intervention. Annu. Rev. Pharmacol. Toxicol. 35: 55-91 [Medline]. |
11. | Lövkvist-Wallström, E., L. Stjernborg-Ulvsbäck, I.E. Scheffler, and L. Persson. 1995. Regulation of mammalian ornithine decarboxylase. Studies on the induction of the enzyme by hypotonic stress. Eur. J. Biochem. 231: 40-44 [Abstract]. |
12. | Pegg, A.E., L.M. Shantz, and C.S. Coleman. 1994. Ornithine decarboxylase: structure, function and translational regulation. Biochem. Soc. Trans. 22: 846-852 [Medline]. |
13. | Paschen, W.. 1992. Polyamine metabolism in different pathological states of the brain. Mol. Chem. Neuropathol. 16: 241-271 [Medline]. |
14. | Traynelis, S.F., M. Hartley, and S.F. Heinemann. 1995. Control of proton sensitivity of the NMDA receptor by RNA splicing and polyamines. Science. 268: 873-876 [Medline]. |
15. | Traynelis, S.F., and S.G. Cull-Candy. 1991. Pharmacological properties and H+ sensitivity of excitatory amino acid receptor channels in rat cerebellar granule neurones. J. Physiol. (Lond.). 433: 727-763 [Abstract]. |
16. | Sullivan, J.M., S.F. Traynelis, H.S. Chen, W. Escobar, S.F. Heinemann, and S.A. Lipton. 1994. Identification of two cysteine residues that are required for redox modulation of the NMDA subtype of glutamate receptor. Neuron. 13: 929-936 [Medline]. |
17. | Fahey, J.M., G.A. Pritchard, and L.G. Miller. 1993. Polyamine neurotoxicity is antagonized by dizocilpine in cultured chick cortical neurons. Neurosci. Lett. 161: 109-112 [Medline]. |
18. | 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 [Medline]. |
19. | Paschen, W.. 1992. Polyamine metabolism in reversible cerebral ischemia. Cerebrovasc. Brain Metab. Rev. 4: 59-88 [Medline]. |
20. | Morgan, D.M.L. 1989. Polyamine oxidases and oxidized polyamines. In The Physiology of Polyamines. U. Bachrach and Y.M. Heimer, editors. CRC Publications, Cleveland, OH. 203-229. |
21. | Paschen, W., J. Hallmayer, and G. Rohn. 1988. Relationship between putrescine content and density of ischemic cell damage in the brain of mongolian gerbils: effect of nimodipine and barbiturate. Acta Neuropathol. 76: 388-394 [Medline]. |
22. | Seiler, N., and F.N. Bolkenius. 1985. Polyamine reutilization and turnover in brain. Neurochem. Res. 10: 529-544 [Medline]. |
23. | Seiler, N., N. Bolkenius, and O.M. Rennert. 1981. Interconversion, catabolism and elimination of the polyamines. Med. Biol. 59: 334-346 [Medline]. |
24. | Bolkenius, F.N., and N. Seiler. 1986. Developmental aspects of polyamine interconversion in rat brain. Int. J. Dev. Neurosci. 4: 217-224 [Medline]. |
25. | Bolkenius, F.N., P. Bey, and N. Seiler. 1985. Specific inhibition of polyamine oxidase in vivo is a method for the elucidation of its physiological role. Biochim. Biophys. Acta. 838: 69-76 [Medline]. |
26. | Seiler, N. 1995. Polyamine oxidase, properties and functions. Prog. Brain Res. 333-344. |
27. | Morgan, D.M.L.. 1987. Polyamines. Essays Biochem. 23: 82-115 [Medline]. |
28. | Houen, G., K. Bock, and A.L. Jensen. 1994. HPLC and NMR investigation of the serum amine oxidase catalyzed oxidation of polyamines. Acta Chem. Scand. 48: 52-60 [Medline]. |
29. | Bouzyk, E., and O. Rosiek. 1988. Clastogenic and cytotoxic effects of spermine oxidation products in mouse lymphoma L5178Y cells. Cancer Lett. 39: 93-99 [Medline]. |
30. | Brunton, V.G., M.H. Grant, and H.M. Wallace. 1994. Spermine toxicity in BHK-21/C13 cells in the presence of bovine serum: the effect of aminoguanidine. Toxicol. In Vitro. 8: 337-341 . |
31. | Gaugas, J.M., and D.L. Dewey. 1978. Evidence for serum binding of oxidized spermine and its potent G1-phase inhibition of cell proliferation. Br. J. Cancer. 39: 548-557 . |
32. | Morgan, D.M.L., U. Bachrach, G. Assaraf, E. Harri, and J. Golenser. 1986. The effect of purified aminoaldehydes produced by polyamine oxidation on the development in vitro of Plasmodium falciparum in normal and glucose-6-phosphate-dehydrogenase-deficient erythrocytes. J. Biochem. 236: 97-101 . |
33. |
Ferrante, A.,
C.M. Rzepczyk, and
A.J. Saul.
1984.
Polyamine
oxidase-mediated trypanosome killing: the role of hydrogen
peroxide and aldehydes.
J. Immunol.
133:
2157-2162
|
34. | Parchment, R.E., and G.B. Pierce. 1989. Polyamine oxidation, programmed cell death, and regulation of melanoma in the murine embryonic limb. Cancer Res. 49: 6680-6686 [Abstract]. |
35. | Kurihara, H., S. Matsuzaki, H. Yamazaki, T. Tsukahara, and M. Tamura. 1993. Relation between tissue polyamine levels and malignancy in primary brain tumors. Neurosurgery (Baltim.). 32: 372-375 [Medline]. |
36. | Gahl, W.A., and H.C. Pitot. 1978. Reversal by aminoguanidine of the inhibition of proliferation of human fibroblasts by spermidine and spermine. Chem.-Biol. Interact. 22: 91-98 [Medline]. |
37. | Henle, K.J., A.J. Moss, and W.A. Nagle. 1986. Mechanism of spermidine cytotoxicity at 37 degrees C and 43 degrees C in Chinese hamster ovary cells. Cancer Res. 46: 175-182 [Abstract]. |
38. | Milani, D., D. Guidolin, L. Facci, T. Pozzan, M. Buso, A. Leon, and S.D. Skaper. 1991. Excitatory amino acid-induced alterations of cytoplasmic free Ca2+ in individual cerebellar granule neurons: role in neurotoxicity. J. Neurosci. Res. 28: 434-441 [Medline]. |
39. |
Zimmerman, G.A.,
M. Meistrell III,
O. Bloom,
K.M. Cockroft,
M. Bianchi,
D. Risucci,
J. Broome,
P. Farmer,
A. Cerami,
H. Vlassara, and
K.J. Tracey.
1995.
Neurotoxicity of
advanced glycation endproducts during focal stroke, and neuroprotective effects of aminoguanidine.
Proc. Natl. Acad. Sci.
USA.
92:
3744-3748
|
40. |
Cockroft, K.M.,
M. Meistrell III,
G.A. Zimmerman,
D. Risucci,
O. Bloom,
A. Cerami, and
K.J. Tracey.
1996.
Cerebroprotective effects of aminoguanidine in a rodent model of
stroke.
Stroke.
27:
1393-1398
|
41. | Bederson, J.B., L.H. Pitts, S.M. Germano, M.C. Nishimura, R.L. Davis, and H.M. Bartkowski. 1986. Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke. 17: 1304-1308 [Abstract]. |
42. | Whitmore, W.L., and T.A. Slotkin. 1985. A simplified method for isocratic HPLC analysis of polyamines. Experientia (Basel). 41: 1209-1211 . |
43. | Bachrach, U., and B. Reches. 1966. Enzymatic assay for spermine and spermidine. Anal. Biochem. 17: 38-48 [Medline]. |
44. | Dickinson, R., and N. Jacobsen. 1970. A new and sensitive test for detection of aldehydes: formation of 6-mercapto-3-substituted-s-triazolo(4,3-b-)-s-tetrazines. Chem. Commun. 1719-1720. |
45. | Ponten, J., and E.H. MacIntyre. 1968. Long term culture of normal and neoplastic human glia. Acta Pathol. Microbiol. Scand. 74: 465-486 [Medline]. |
46. | Bluestein, H.G.. 1978. Neurocytotoxic antibodies in serum of patients with systemic lupus erythematosus. Proc. Natl. Acad. Sci. USA. 75: 3965-3969 [Abstract]. |
47. | Sieuwerts, A.M., J.G. Klijn, H.A. Peters, and J.A. Foekens. 1995. The MTT tetrazolium salt assay scrutinized: how to use this assay reliably to measure metabolic activity of cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell survival. Eur. J. Clin. Chem. Clin. Biochem. 33: 813-823 [Medline]. |
48. | Meistrell, M.E. III, G.I. Botchkina, H. Wang, E. Di Santo, K.M. Cockroft, O. Bloom, J.M. Vishnubhakat, P. Ghezzi, and K.J. Tracey. 1997. Tumor necrosis factor is a brain-damaging cytokine in cerebral ischemia. Shock. 8: 341-348 [Medline]. |
49. | Holtta, E.. 1977. Oxidation of spermidine and spermine in rat liver: purification and properties of polyamine oxidase. Biochemistry. 16: 91-100 [Medline]. |
50. |
Flayeh, K.A..
1988.
Spermidine oxidase activity in serum of
normal and schizophrenic subjects.
Clin. Chem.
34:
401-403
|
51. | Gahl, W.A., and H.C. Pitot. 1982. Polyamine degradation in foetal and adult bovine serum. Biochem. J. 202: 603-611 [Medline]. |
52. | Seiler, N.. 1990. Polyamine metabolism. Digestion. 46: 319-330 [Medline]. |
53. | Gavrieli, Y., Y. Sherman, and S.A. Ben-Sasson. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell. Biol. 119: 493-501 [Abstract]. |
54. | Furuya, Y., S. Ohta, and H. Ito. 1997. Apoptosis of androgen-independent mammary and prostate cell lines induced by topoisomerase inhibitors: common pathway of gene regulation. Anticancer Res. 17: 2089-2093 [Medline]. |
55. |
Bhat, R.V.,
R. DiRocco,
V.R. Marcy,
D.G. Flood,
Y. Zhu,
P. Dobrzanski,
R. Siman,
R. Scott,
P.C. Contreras, and
M. Miller.
1996.
Increased expression of IL-1beta converting enzyme in hippocampus after ischemia: selective localization in
microglia.
J. Neurosci.
16:
4146-4154
|
56. | Loddick, S.A., A. MacKenzie, and N.J. Rothwell. 1996. An ICE inhibitor, z-VAD-DCB attenuates ischaemic brain damage in the rat. Neuroreport. 7: 1465-1468 [Medline]. |
57. |
Hara, H.,
R.M. Friedlander,
V. Gagliardini,
C. Ayata,
K. Fink,
Z. Huang,
M. Shimizu-Sasamata,
J. Yuan, and
M.A. Moskowitz.
1997.
Inhibition of interleukin 1beta converting
enzyme family proteases reduces ischemic and excitotoxic
neuronal damage.
Proc. Natl. Acad. Sci. USA.
94:
2007-2012
|
58. |
Friedlander, R.M.,
V. Gagliardini,
H. Hara,
K.B. Fink,
W. Li,
G. MacDonald,
M.C. Fishman,
A.H. Greenberg,
M.A. Moskowitz, and
J. Yuan.
1997.
Expression of a dominant
negative mutant of interleukin-1![]() |
59. | Gillardon, F., B. Bottiger, B. Schmitz, M. Zimmermann, and K.A. Hossmann. 1997. Activation of CPP-32 protease in hippocampal neurons following ischemia and epilepsy. Brain Res. Mol. Brain Res. 50: 16-22 [Medline]. |
60. |
Zhang, F.,
R.M. Casey,
E. Ross, and
C. Iadecola.
1996.
Aminoguanidine ameliorates and L-arginine worsens brain
damage from intraluminal middle cerebral artery occlusion.
Stroke.
27:
317-323
|
61. | Gilad, G.M., V.H. Gilad, and R.J. Wyatt. 1993. Accumulation of exogenous polyamines in gerbil brain after ischemia. Mol. Chem. Neuropathol. 18: 197-210 [Medline]. |
62. | Harada, J., and M. Sugimoto. 1997. Polyamines prevent apoptotic cell death in cultured cerebellar granule neurons. Brain Res. 753: 251-259 [Medline]. |
63. | Xie, X., M.E. Tome, and E.W. Gerner. 1997. Loss of intracellular putrescine pool-size regulation induces apoptosis. Exp. Cell Res. 230: 386-392 [Medline]. |
64. |
Casero, R.A. Jr., and
A.E. Pegg.
1993.
Spermidine/spermine
N1-acetyltransferase![]() |
65. | Tipnis, U.R., G.Y. He, and M.F. Khan. 1997. Differential induction of polyamine oxidase activity in liver and heart of iron-overloaded rats. J. Toxicol. Environ. Health. 51: 235-244 [Medline]. |
66. | Li, L., R.F. Hamilton Jr., D.E. Taylor, and A. Holian. 1997. Acrolein-induced cell death in human alveolar macrophages. Toxicol. Appl. Pharmacol. 145: 331-339 [Medline]. |
67. | Fernandez, C., R.M. Sharrard, M. Talbot, B.D. Reed, and N. Monks. 1995. Evaluation of the significance of polyamines and their oxidases in the aetiology of human cervical carcinoma. Br. J. Cancer. 72: 1194-1199 [Medline]. |
68. | Coyle, J.T., and P. Puttfarcken. 1993. Oxidative stress, glutamate, and neurodegenerative disorders. Science. 262: 689-695 [Medline]. |
69. | Rothwell, N.J., and P.J. Strijbos. 1995. Cytokines in neurodegeneration and repair. Int. J. Dev. Neurosci. 13: 179-185 [Medline]. |
70. | Irikura, K., P.L. Huang, J. Ma, W.S. Lee, T. Dalkara, M.C. Fishman, T.M. Dawson, S.H. Snyder, and M.A. Moskowitz. 1995. Cerebrovascular alterations in mice lacking neuronal nitric oxide synthase gene expression. Proc. Natl. Acad. Sci. USA. 92: 6823-6827 [Abstract]. |
71. | Rothwell, N.J., and J.K. Relton. 1993. Involvement of interleukin-1 and lipocortin-1 in ischaemic brain damage. Cerebrovasc. Brain Metab. Rev. 5: 178-198 [Medline]. |
72. | Taupin, V., S. Toulmond, A. Serrano, J. Benavides, and F. Zavala. 1993. Increase in IL-6, IL-1 and TNF levels in rat brain following traumatic lesion. Influence of pre- and post-traumatic treatment with Ro5 4864, a peripheral-type (p site) benzodiazepine ligand. J. Neuroimmunol. 42: 177-185 [Medline]. |
73. | Saito, K., K. Suyama, K. Nishida, Y. Sei, and A.S. Basile. 1996. Early increases in TNF-alpha, IL-6 and IL-1 beta levels following transient cerebral ischemia in gerbil brain. Neurosci. Lett. 206: 149-152 [Medline]. |
74. | Choi, D.W.. 1992. Excitotoxic cell death. J. Neurobiol. 23: 1261-1276 [Medline]. |
75. | Montague, P.R., C.D. Gancayco, M.J. Winn, R.B. Marchase, and J.J. Friedlander. 1994. Role of NO production in NMDA receptor-mediated neurotransmitter release in cerebral cortex. Science. 263: 973-976 [Medline]. |
76. | Hara, H., K. Fink, M. Endres, R.M. Friedlander, V. Gagliardini, J. Yuan, and M.A. Moskowitz. 1997. Attenuation of transient focal cerebral ischemic injury in transgenic mice expressing a mutant ICE inhibitory protein. J. Cereb. Blood Flow Metab. 17: 370-375 [Medline]. |
77. |
Zhang, M.,
T. Caragine,
H. Wang,
P.S. Cohen,
G. Botchkina,
K. Soda,
M. Bianchi,
P. Ulrich,
A. Cerami,
B. Sherry, and
K.J. Tracey.
1997.
Spermine inhibits proinflammatory
cytokine synthesis in human mononuclear cells: a counterregulatory mechanism that restrains the immune response.
J.
Exp. Med.
185:
1-10
|
78. | Seiler, N., F.N. Bolkenius, B. Knodgen, and P. Mamont. 1980. Polyamine oxidase in rat tissues. Biochim. Biophys. Acta. 615: 480-488 [Medline]. |