Kainate-induced seizure activity stimulates the polyamine interconversion pathway in rat brain
Michel Baudry*, Imad Najm
Neuroscience Program, HNB 311, University of Southern California, Los Angeles, CA 90089-2520, USA
Received 29 September 1993; Revised version 27 December 1993; Accepted 16 February 1994
Abstract Systemic injection of kainic acid in adult rat is accompanied by a large increase in the accumulation of acetylated derivatives of
spermidine and spermine in the hippocampus and piriform cortex of animals pretreated with the polyamine oxidase inhibitor. M D L 72527. Furthermore, the activity of the enzyme spermine/spermidine acetyltransferase is increased at 8 and 16 h after kainate injection in piriform cortex and hippocampus. These results indicate that the polyamine interconversion pathway is rapidly activated in limbic areas following kainate-induced seizure activity, and suggest that this pathway might participate in the resulting neuronal damage.
Numerous reports have shown that polyamine metab- olism in brain is rapidly increased in response to a variety of stimuli, including ischemia, seizure activity or me- chanical injury [2,12,18,23]. Under all these conditions, the expression and activity of the rate-limiting enzyme in polyamine metabolism, ornithine decarboxylase (ODC), are increased resulting in elevation of putrescine levels. While the synthetic pathway and its regulation have been studied in great detail, much less is known concerning the interconversion pathway which has been described by Seiler and colleagues over the last 10 years [4,26]. This pathway involves the N-acetylation of polyamines medi- ated by spermine/spermidine N-acetyltransferase (SAT) followed by the action of a polyamine oxidase (PAO) which converts N-acetylspermine and N-acetylspermid- ine into spermidine and putrescine, respectively [26]. An important by-product of this pathway is hydrogen per- oxide which has often been implicated in cell damage [1 ], and it has thus been proposed that activation of this pathway might participate in programmed cell death in embryo [9]. Several toxic stimuli produce a rapid activa- tion of SAT in mammalian cells and peripheral tissues [5,7,15,16] but very few studies have examined changes
in polyamine acetylation in brain [20]. Kainate (KA)- induced seizure activity produces marked changes in ODC activity and in polyamine levels in hippocampus [10,18] and increased putrescine levels have been pro- posed to be a marker for neuronal pathology resulting from seizure activity and ischemia [18,19,21,22]. As pu- trescine can be produced by both the synthetic pathway and the interconversion pathway, it is important to de- termine the relative contribution of each pathway. Po- tent inhibitors of PAO have been developed and shown to produce accumulation of acetylated polyamines in various tissues including brain, thus providing an indi- rect measure of polyamine turn-over through the acetyl- ation pathway [3]. In the present study we used this approach as well as the direct measure of SAT activity to evaluate the changes in polyamine acetylation follow- ing KA-induced seizure activity.
Male Sprague-Dawley rats (150 200 g) were kept on a 12 h light cycle and had access to food and water ad libitum. KA (10 mg/kg) was injected subcutaneously. The polyamine oxidase inhibitor MDL 72527 was kindly provided by Dr. Seiler from Merell Dow Laboratories and was administered i.p. at 50 mg/kg, 24 h before KA injection and again 1 h before KA injection. Animals were sacrificed at various time intervals after KA- induced seizure activity. No difference in the latency,
duration and intensity of seizure activity elicited by KA administration was observed between MDL 72527- treated animals and saline-treated animals.
For the determination of ODC and SAT activity, brains were rapidly dissected, and various brain struc.- tures were homogenized in 5 volumes of an homogeniza-- tion buffer (buffer A) consisting of 10 mM Tris-HC1, pH 7.4, 0.32 M sucrose, 2 mM EDTA, 1 mM EGTA, 0.1 mM leupepiin and 1/tg/ml TPC K. Aliquots of homoge- nares were dihited in either ODC buffer (150 mM Tris- HC1, pH 7.4, 0.6 ntM EDTA, 30 mM dithiothreitol, and 0.6 mM pyridoxal-5-phosphate) or SAT buffer (10 mM Tris-HC1, 0 1 mM EDTA, 2.5 mM dithiothreitol). ODC activity was measured by determining the amount of HCO, released fl'om [HC]ornithine its previously de- scribed [2]. SAT activity was measured by determining the conversion of [~H]acetyl-CoA into [~H]N-acetylsper- midine according to Matsui et al. [I 5] with minor modi- fications. SAT activity was defined its the difference in [-~H]acetylspermidine formed in the presence and absence of 3 mM spermidine. The incubation was performed at 30 °C l\~r 1{) min (final vohune, 100 #1) in the presence of 0.1 mM pargylipe and was stopped by the addition of 20 #1 of 1 mM NH4OCI and boiling of the sltmples for 5 min. ['H]Acetylspermidine wits then separated fiorn [3H]acetyl-(oA by filtration on cellulose phosphate fil- ters (P 81, Whatman) and washing of the filters in 3 baths of H~O and two baths of EtOH.
Polyamines and their N-acelyl derivatives were as- sayed by using the dansylation method described by' De- siderio et al. [11], which consists in pre-derivatization of perchlorate extracts of tissues followed by separation with HPLC. Without MDL 72527 pretreatment, levels of N-acetylspermidine and N-acetylspermine were below or at the lilnit of detection of our techniques, whereas with such a pretreatment peaks corresponding to authen- tic acetylated derivatives were reliably observed.
Analysis of variance was used to evaluate statistical significance between groups.
Polyamines and their N-acetyl derivatives were meas- ured in hippocampus and piriform cortex 16 h after KA- induced seizure activity in animals pretreated with saline or with the PAO inhibitor, MDL 72527 (Fig. 1). Pre- treatment with MDL 72527 alone produced, as expected, the accuinulation of N-acetylspermine and N-acetyl- spermidinc whicla could not be detected in control ani- mals. In agreernent with previously published data [10,18]. KA treatment alone produced large increases in putrescinc k'vels in hippocanlpus and piriform cortex, its well its significant decreases in spermidine and spermine levels in piril\~rm cortex. While N-acetylspermidine was not detectable, very 1o~' levels of N-acetylspermine were measured under these conditions. KA treatment of ani- mals pre-treated with M DL 72527 produced significantly larger increases in N-acetylspermidine and N-acetylsper- mine levels than in MDL 72527-treated animals. In addi-
O
3130
200"
100"
[] Control [] MDL
[] KA [] MDL+KA
ni N-Ac-Spd
Hippocampus
Putrescine N-Ac-Spn Spermidine Spermlne
E
300
200
100
Piriform Cortex
[ ] Control
[] MDL [] KA [] MDL+KA
N-Ac-Spd
t *
Putrescine N-Ac-Spn Spermidine Spermme
Fig. 1. Levels of polyamines and acetylated derivatives in hippocampus and piriform cortex following KA-induced seizure activity. Groups of rats were treated with saline (Control), M DL 72527 (MDL), kainic acid (KA) or both (MDL + KA) as described in the text and were sacrificed 16 h after KA rejection. Polyamines (putrescine, spernaidine, and sper- mine) and acetyl deriw~tives (N-acetylspermidine (N-Ac-Spd) and N- acctylspermine (N-Ac-Spn)) wcre analyzed by HPLC in hippocampus (top panel) and piriform cortex (bonom panel) :is described in the text. Results are expressed as nmol/g tissue and are means _+ S.E.M. of 3 animals (control and MDL) or 6 animals (KA and KA + MDL). The absence of a bar in some cases reflects the undelectability of the com- pounds.*P < 0.05 as compared to MDL alone: 'P < 0.05 as compared to KA alone: 'P < 0.05 :is compared to colln-ol (ANOVA, post-hoe test).
tion, MDL 72527 pretreatment significantly reduced the increase in putrescine levels elicited by KA treatment. It also significantly reduced the decrease in spermine levels in piriform cortex. In order to evaluate the increased turn-over of spermine and spermidine through the inter- conversion pathway, we calculated the ratios N-ace- tylspermidine over spermidine and N-acetylspermine over spermine in saline- and KA-treated rats (both groups being pre-treated with MDL 72527) (as the ratios were very similar m hippocampus and piril\~rm cortex, the averaged values for each structure were used for each animal). Interestingly, the ratio was 1{) 15 times higher
M. Baudry, 1. Najm/Neuroscience Letters 171 (1994) 151 154 153
for spermidine than for spermine in both control (0.46 + 0.03 vs. 0.031 + 0.007) and KA-treated animals (2.54 + 0.22 vs. 0.16 + 0.04, respectively: means + S.E.M. of 3 experiments in control and 6 experiments in KA-treated animals). Thus, KA-induced seizure activ- ity resulted in a 5-fold increase in both ratios, clearly indicating an increase in the rate of polyamine acetyla- tion.
This notion was further confirmed by measuring SAT activity in piriform cortex at different time intervals fol- lowing KA-induced seizure activity (Fig. 2). Significant increases in SAT activity were found at 8 and 16 h fol- lowing KA-induced seizure activity. By 24 h, SAT activ- ity was almost back to control values. The increase in SAT activity was about 4-fold, a value close to that obtained for the ratio between acetylated polyamines and their respective des-acetylated form. The increase in SAT activity was much lower than that observed for ODC activity, although it appeared to follow a similar time-course. Similar results were observed in hippocam- pus (data not shown).
The present results clearly indicate that KA-induced seizure activity is associated with a large stimulation of the polyamine interconversion pathway. This was evi- denced both by an increase in SAT activity, as well as in acetylated polyamine levels following the inhibition of polyamine oxidase. Under such conditions, the ratios N-acetylspermidine over spermidine and N-acetylsper- mine over spermine provide an index of the in vivo rate of polyamine acetylation [3]. Both approaches provided a similar estimate of the increased rate of acetylation by a factor of about 4-5. The results suggest that the in- crease in putrescine levels reported by us [18,19] and others [10,22] following various types of insults are due not only to the increased expression of ODC and the stimulation of the synthetic pathway but also to the stim- ulation of the interconversion pathway (as shown in Fig. 1, pretreatment of the animals with an inhibitor of poly- amine oxidase significantly reduced the increase in pu- trescine elicited by KA treatment). As the interconver- sion pathway generates H202 it is conceivable that acti- vation of this pathway contributes to the pathological manifestations of seizure activity, such as neuronal dana- age and glial proliferation, as oxygen radicals have been suggested to be involved in excitotoxicity [8,17]. How- ever, in preliminary studies we did not observe a signifi- cant protective effect of M D L 72527 pretreatment on KA-induced neuronal damage, as evaluated with mark- ers of glial proliferation or spectrin breakdown (unpub- lished obserwltions). However, this does not exclude a role for the activation of the interconversion pathway in excitotoxicity. In particular, intracellular polyamines are presumed to be mostly bound and acetylation might pro- vide a means by which polyamines could be released extracellularly [7,26]. It has recently been shown that activation of N M D A receptors is potentiated by polyam-
Fig. 2. SAT and ODC activity in piriform cortex at various times alter KA injection. Groups of 3-6 rats were injected with saline or KA (10 mg/kg; s.c.) and were sacrificed after the indicated survival times. SAT and ODC activities were measured in piriform cortex as described under Methods. Results are expressed in fmol/mg protein& or pmol/mg protein/h for SAT or ODC, respectively, and are means _+ S.E.M. *P < 0.05 as compared to control (ANOVA. post-hoc testL
ines acting on an extracellular site of the receptors [27] and therefore increased acetylation might participate in- directly to neuronal damage by stimulating N M D A re- ceptors, an event which has been implicated in several forms of neuronal damage [24]. In agreement with this idea, spermine levels were significantly decreased in hip- pocampus and piriform cortex following KA-induced seizure activity. Furthermore, increased levels of poly- amines have been found in extracellular fluids by micro- dialysis studies following N M D A receptor activation [13]. Spermine and spermidine have also been shown to produce neuronal damage independently of their effect on N M D A receptors [6]. In particular it has been shown that polyamines interact with calcium channels [25] and with calcium homeostasis mechanisms [14]. Further work will be needed to more precisely establish the con- tribution of the polyamine interconversion pathway in the pathological consequences of KA-induced seizure ac- tivity.
This work was supported by Grant BNS 18427 from N I N D S to M.B.
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