Life Sciences, Vol. Printed in the U S A
51, pp. PL 77-81
Pergamon
PHARMACOLOGY Accelerated
Press
LETTERS
Communication
E X C I T O T O X I N I N D U C T I O N OF O R N I T H I N E D E C A R B O X Y L A S E IN C E R E B R A L C O R T E X IS R E D U C E D BY P H O S P H O L I P A S E A2 I N H I B I T I O N
I. M. Gardiner, Andy Li, Nikinje Patel, Stephen Ball and J. de Belleroche* Department of Biochemistry, Chining Cross & Westminster Medical School, Fulham Palace Road, London W6 8RF (Submitted April 15, 1992; accepted May 6, 1992; received in final form June 16, 1992)
Abstract. The enzyme ornithine decarboxylase (ODC) has been shown to be induced by a number of conditions such as cold-injury, kindling, ischaemia and excitotoxin injection. In previous studies we have characterised the cortical response to kainate injection into the nucleus basalis and shown a substantial increase in both ODC mRNA and enzyme activity which reaches a maximum at 8h. This response is completely prevented by treatment with MK-801, indicating the involvement of NMDA receptors in mediating this response. Whilst NMDA receptors are known to gate a cation channel leading to increased calcium entry, an additional effect on the release of arachidonic acid has been reported. The possibility that NMDA receptor mediated activation of phospholipase A2 and release of arachidonic acid might mediate this ODC response was investigated in this study by treatment with the phospholipase inhibitors quinacrine and dexamethasone. Treatment of animals with quinacrine (100 mg/kg) at the time of injection of kainate into the nucleus basalis caused a significant attenuation of the induction of ODC in cerebral cortex of 43%. No further attenuation was seen at higher doses. A similar reduction in ODC induction was seen after treatment with dexamethasone (1 mg/kg) but a greater effect could be obtained (65% attenuation) at higher doses. The possible involvement of arachidonic acid derivatives in mediating ODC induction was further investigated by treatment with the cyclo-oxygenase inhibitor indomethacin and the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA). Indomethacin was able to significantly attenuate the induction of ODC (> 60%) whilst NDGA (30 mg/kg) was ineffective. These results indicate the possible role of arachidonic acid derivatives in the regulation of the expression of ODC in cerebral cortex after excitotoxin injection.
Introduction Glutamate has recently been implicated as the key agent mediating neurotoxic events following ischaemic episodes. The glutamate analogue domoic acid has been shown to cause encephalopathy whose pathology and consequential intellectual impairment reflect the specific distribution of the kainate subtype of glutamate receptor (1). The precise mechanism whereby excess glutamate arising from compromised cells causes neuronal death is unknown, although elevated intracellular calcium ions and free radical production have been strongly indicated as mediators. A further event associated with a range of injury responses, e.g. ischaemia, cold-injury, excitotoxin lesion and mechanical trauma, is the induction of the enzyme ornithine decarboxylase (ODC: 2-9). * Correspondence to: Dr. J. de Belleroche, Deparmmnt of Biochemistry, Charing Cross and Westminster Medical School, Fulham Palace Road, London, W6 8RF
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
PL-78
Excitotoxin
and ODC
in C e r e b r a l
Cortex
Vol.
51, No.
8, 1992
The significance of this response in mediating neurotoxicity is demonstrated by the fact that Nmethyl-D-aspartate (NMDA)-mediated neurotoxicity of mouse cortical cells can be completely prevented by co-exposure to the irreversible ODC inhibitor difluoromethyl omithine (DFMO: 10). We have previously characterised the induction of ODC mRNA and enzyme activity in rat cerebral cortex following unilateral injection of kainate into the nucleus basalis (9,11). The induction is associated with a persistent excitation which is dependent on the involvement of an NMDA receptor as ODC induction can be completely prevented by treatment with the NMDA antagonist, MK-801. Induction of ODC mRNA is preceded by the induction of mRNA for two immediate early genes, c-fos and c-jun (12,13). The mechanism whereby NMDA receptors could mediate the induction of ODC and other genes has been further elucidated in this study by investigating the role of NMDA receptor-stimulated arachidonic acid release in the induction process. NMDA receptor-mediated release of arachidonic acid was first demonstrated in primary cultures of cerebellar granule cells (14) and mouse striatal neurones (15) and has subsequently been demonstrated in other tissues. The release of arachidonic acid has been shown to be dependent on the presence of extracellular calcium and involve the action of phospholipase A 2, as the phospholipase A 2 inhibitor quinacrine is able to prevent arachidonic acid release (16). In this study we have investigated the effects of quinacrine and dexamethasone on the induction of ODC and further explored the possible involvement of arachidonic acid derived eicosanoids on the induction process by use of indomethacin and nordihydroguaiaretic acid (NDGA) to inhibit cyclo-oxygenase and lipoxygenase respectively and hence the generation of prostaglandins and leukotrienes. Methods Lesion of the nucleus basalis Unilateral lesion of the rat nucleus basalis was carried out as previously characterised by de Belleroche et al (17). Male CFY rats (240-280 g at time of surgery) were used for all experiments. Rats were anaesthetised with pentobarbital (Sagatal; May and Baker) and placed in a stereotactic apparatus. Unilateral lesion of the right nucleus basalis was carried out by injection of kainate (0.5 ~1 of a 2 mg/ml solution in water, pH 7.0) into the nucleus basalis (coordinates: AP +1.2, lateral -2.7 with respect to bregma; incisor bar at 5 mm above the intraaural line; depth 8.8 mm below the skull surface). Injection was made over a 60-s period and the needle was left in place for 5 rain to prevent reflux of excitotoxin. All drugs were administered i.p. at the time of lesion. Assay of ODC activity At 8 h after lesion, ipsilateral and contralateral frontoparietal cortex (rostral cerebral cortex limited medially by the boundary with cingulate cortex, laterally by the rhinal fissure, and caudally by the boundary with occipital lobe) were dissected out, frozen in liquid nitrogen and stored at -70°C. For assay of ODC activity, tissue samples were homogenised in cold 25 mM Tris-HC1 (pH 7.4) containing 0.1 mM EDTA, 5 mM dithiothreitol, and 0.1 mM pyridoxal 5'-phosphate. Homogenates were centriftlged at 20,000 g for 25 rain at 4°C, and the supernatants were assayed for ODC activity according to the procedure of Baudry et al (2) by measuring the release of 14CO2 from [1-14C] ornithine (final concentration, 50 ~M; 5 ~Ci/~mol). Incubations were carried out for 1 h at 37°C, and the protein concentration of each sample was determined by the method of Bradford (18). Results are expressed as picomoles of 14CO2 released/rag of protein/h. Resnlts Effect of phospholipase A 2 inhibition on the induction of ODC by kainate. Injection of kainate into the nucleus basalis causes a massive 200-fold induction of ODC in ipsilateral cerebral cortex which is maximal at 8 hours and can be completely prevented by MK-801 treatment (lmg/kg) (9). A relatively minor increase is seen in the contralateral cortex. This response was used to investigate the involvement of phospholipase A2 activity in the induction of ODC. Two drug tre:ttments were used initially to test this hypothesis, quinacrine, which directly
Vol.
51, No.
8, 1992
Excitotoxin
and O D C in C e r e b r a l
Cortex
PL-79
inhibits phospholipase A2 by interfering with the fom~ation of the enzyme substrate complex, and dexamethasone, which reduces arachidonic acid release through the intermediary action o f lipocortin. Quinacrine at 100 mg/kg given at the time of lesion caused a significant (p < 0.005) attenuation of the induction of ODC (41%) in ipsilateral cortex. A similar attenuation was seen at 200 mg/kg quinacrine, (Fig. 1). A much smaller induction of ODC was seen in cerebral cortex
"* 800
p< 0.05 p< 0.005
7
c::
>
T
E O
T
600
(,9 400
o
I
I 200
,-,---IT
*
qulnacrine 0 100 200 mg/kg
indomethacin 0 30 60 rag/ kg
NDGA 0
30
mg/kg
Fig. 1.
Effect o f quinacrine, indomethacin and NDGA on induction of ODC in cerebral cortex by unilateral injection of kainate. A unilateral injection of kainate was made into the nucleus basalis and cerebral cortex was removed at 8 hours from the ipsilateral and contralateral hemispheres. Animals were treated with either vehicle or drug at the time of lesion. Vehicles used were water for quinacrine, and 1 M NazCO3 for indomethacin and NDGA. Values are means (picomoles 14CO2 released/rag protein/h) with the SEMs shown by error bars. Values in the contralateral cortex are shown as hatched bars superimposed on clear histograms for the ipsilateral cerebral cortex. The number of animals in each group was 6-10. The asterisks indicate the level of significance when comparing drug treated groups with vehicle treated groups. Students' t test (tmpaired). contralateral to the kainate injection but this was only inhibited by quinacrine at 200 mg/kg. Treatment with dexamethasone at the time of lesion produced a dose dependent attenuation in the response, which was significant at doses of 1 mg/kg and above. The greatest attenuation in response was seen at a dose of 2 mg/kg where the response was reduced by 67% in dexamethasone-treated animals compared to vehicle treated animals (Fig. 2). The induction of ODC in contralateral cortex was also significantly reduced at this dose of dexamethasone but not at other doses. Effects of cyclo-oxygenase and lipoxygenase inhibitors on the induction of ODC. The involvement of prostaglandins or leukotrienes in mediating the induction of ODC was investigated by the use of indomethacin and NDGA. Administration of indomethacin at the time of lesion caused a significant attenuation of the induction of ODC in cerebral cortex ipsilateral to the kainate injection. This effect was significant at a dose of 30 mg/kg where an inhibition of 38% was obtained. The induction o f ODC was further attenuated (58%) at 60 mg/kg (Fig.l). The lipoxygenase inhibitor NDGA was without effect on the induction of ODC up to a dose of 30 mg/kg. It was not possible to use a higher dose of this agent due to its poor solubility.
PL-80
Excitotoxin
700 ._Cl (1)
and O D C in C e r e b r a l
Cortex
Vol.
51, No.
8, 1992
o-- c o n t r a l a t e r a l ipsilateral
~
600
500
o
O_
O
O E3% O E
400
Ob
O_
300
200
v
100 0
I
0
. . . . . . . .
I
. . . . . . . .
.01
I
. . . . . . . .
.1
I
1
. . . . . . . .
I
10
dexamethasone (mg/kg) Fig. 2. Effect of dcxan/elhasone on the induction of ODC by kainate. Animals were treated with vehicle (0) or dexamethasone at a range of concentrations from 0.01 to 3 mg/kg. ODC was measured in ipsilateral or contralateral cereb,'al cortex at 8 hours as described in the Legend to Fig. 1. Values are expressed as Fig. 1. The number of animals used was 6-10 lit each concentration of the drug. The asterisk and double asterisk indicates that the wflues all significantly reduced when compared to the vehicle injected group with a significance of p
51, pp. PL 77-81
Pergamon
PHARMACOLOGY Accelerated
Press
LETTERS
Communication
E X C I T O T O X I N I N D U C T I O N OF O R N I T H I N E D E C A R B O X Y L A S E IN C E R E B R A L C O R T E X IS R E D U C E D BY P H O S P H O L I P A S E A2 I N H I B I T I O N
I. M. Gardiner, Andy Li, Nikinje Patel, Stephen Ball and J. de Belleroche* Department of Biochemistry, Chining Cross & Westminster Medical School, Fulham Palace Road, London W6 8RF (Submitted April 15, 1992; accepted May 6, 1992; received in final form June 16, 1992)
Abstract. The enzyme ornithine decarboxylase (ODC) has been shown to be induced by a number of conditions such as cold-injury, kindling, ischaemia and excitotoxin injection. In previous studies we have characterised the cortical response to kainate injection into the nucleus basalis and shown a substantial increase in both ODC mRNA and enzyme activity which reaches a maximum at 8h. This response is completely prevented by treatment with MK-801, indicating the involvement of NMDA receptors in mediating this response. Whilst NMDA receptors are known to gate a cation channel leading to increased calcium entry, an additional effect on the release of arachidonic acid has been reported. The possibility that NMDA receptor mediated activation of phospholipase A2 and release of arachidonic acid might mediate this ODC response was investigated in this study by treatment with the phospholipase inhibitors quinacrine and dexamethasone. Treatment of animals with quinacrine (100 mg/kg) at the time of injection of kainate into the nucleus basalis caused a significant attenuation of the induction of ODC in cerebral cortex of 43%. No further attenuation was seen at higher doses. A similar reduction in ODC induction was seen after treatment with dexamethasone (1 mg/kg) but a greater effect could be obtained (65% attenuation) at higher doses. The possible involvement of arachidonic acid derivatives in mediating ODC induction was further investigated by treatment with the cyclo-oxygenase inhibitor indomethacin and the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA). Indomethacin was able to significantly attenuate the induction of ODC (> 60%) whilst NDGA (30 mg/kg) was ineffective. These results indicate the possible role of arachidonic acid derivatives in the regulation of the expression of ODC in cerebral cortex after excitotoxin injection.
Introduction Glutamate has recently been implicated as the key agent mediating neurotoxic events following ischaemic episodes. The glutamate analogue domoic acid has been shown to cause encephalopathy whose pathology and consequential intellectual impairment reflect the specific distribution of the kainate subtype of glutamate receptor (1). The precise mechanism whereby excess glutamate arising from compromised cells causes neuronal death is unknown, although elevated intracellular calcium ions and free radical production have been strongly indicated as mediators. A further event associated with a range of injury responses, e.g. ischaemia, cold-injury, excitotoxin lesion and mechanical trauma, is the induction of the enzyme ornithine decarboxylase (ODC: 2-9). * Correspondence to: Dr. J. de Belleroche, Deparmmnt of Biochemistry, Charing Cross and Westminster Medical School, Fulham Palace Road, London, W6 8RF
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
PL-78
Excitotoxin
and ODC
in C e r e b r a l
Cortex
Vol.
51, No.
8, 1992
The significance of this response in mediating neurotoxicity is demonstrated by the fact that Nmethyl-D-aspartate (NMDA)-mediated neurotoxicity of mouse cortical cells can be completely prevented by co-exposure to the irreversible ODC inhibitor difluoromethyl omithine (DFMO: 10). We have previously characterised the induction of ODC mRNA and enzyme activity in rat cerebral cortex following unilateral injection of kainate into the nucleus basalis (9,11). The induction is associated with a persistent excitation which is dependent on the involvement of an NMDA receptor as ODC induction can be completely prevented by treatment with the NMDA antagonist, MK-801. Induction of ODC mRNA is preceded by the induction of mRNA for two immediate early genes, c-fos and c-jun (12,13). The mechanism whereby NMDA receptors could mediate the induction of ODC and other genes has been further elucidated in this study by investigating the role of NMDA receptor-stimulated arachidonic acid release in the induction process. NMDA receptor-mediated release of arachidonic acid was first demonstrated in primary cultures of cerebellar granule cells (14) and mouse striatal neurones (15) and has subsequently been demonstrated in other tissues. The release of arachidonic acid has been shown to be dependent on the presence of extracellular calcium and involve the action of phospholipase A 2, as the phospholipase A 2 inhibitor quinacrine is able to prevent arachidonic acid release (16). In this study we have investigated the effects of quinacrine and dexamethasone on the induction of ODC and further explored the possible involvement of arachidonic acid derived eicosanoids on the induction process by use of indomethacin and nordihydroguaiaretic acid (NDGA) to inhibit cyclo-oxygenase and lipoxygenase respectively and hence the generation of prostaglandins and leukotrienes. Methods Lesion of the nucleus basalis Unilateral lesion of the rat nucleus basalis was carried out as previously characterised by de Belleroche et al (17). Male CFY rats (240-280 g at time of surgery) were used for all experiments. Rats were anaesthetised with pentobarbital (Sagatal; May and Baker) and placed in a stereotactic apparatus. Unilateral lesion of the right nucleus basalis was carried out by injection of kainate (0.5 ~1 of a 2 mg/ml solution in water, pH 7.0) into the nucleus basalis (coordinates: AP +1.2, lateral -2.7 with respect to bregma; incisor bar at 5 mm above the intraaural line; depth 8.8 mm below the skull surface). Injection was made over a 60-s period and the needle was left in place for 5 rain to prevent reflux of excitotoxin. All drugs were administered i.p. at the time of lesion. Assay of ODC activity At 8 h after lesion, ipsilateral and contralateral frontoparietal cortex (rostral cerebral cortex limited medially by the boundary with cingulate cortex, laterally by the rhinal fissure, and caudally by the boundary with occipital lobe) were dissected out, frozen in liquid nitrogen and stored at -70°C. For assay of ODC activity, tissue samples were homogenised in cold 25 mM Tris-HC1 (pH 7.4) containing 0.1 mM EDTA, 5 mM dithiothreitol, and 0.1 mM pyridoxal 5'-phosphate. Homogenates were centriftlged at 20,000 g for 25 rain at 4°C, and the supernatants were assayed for ODC activity according to the procedure of Baudry et al (2) by measuring the release of 14CO2 from [1-14C] ornithine (final concentration, 50 ~M; 5 ~Ci/~mol). Incubations were carried out for 1 h at 37°C, and the protein concentration of each sample was determined by the method of Bradford (18). Results are expressed as picomoles of 14CO2 released/rag of protein/h. Resnlts Effect of phospholipase A 2 inhibition on the induction of ODC by kainate. Injection of kainate into the nucleus basalis causes a massive 200-fold induction of ODC in ipsilateral cerebral cortex which is maximal at 8 hours and can be completely prevented by MK-801 treatment (lmg/kg) (9). A relatively minor increase is seen in the contralateral cortex. This response was used to investigate the involvement of phospholipase A2 activity in the induction of ODC. Two drug tre:ttments were used initially to test this hypothesis, quinacrine, which directly
Vol.
51, No.
8, 1992
Excitotoxin
and O D C in C e r e b r a l
Cortex
PL-79
inhibits phospholipase A2 by interfering with the fom~ation of the enzyme substrate complex, and dexamethasone, which reduces arachidonic acid release through the intermediary action o f lipocortin. Quinacrine at 100 mg/kg given at the time of lesion caused a significant (p < 0.005) attenuation of the induction of ODC (41%) in ipsilateral cortex. A similar attenuation was seen at 200 mg/kg quinacrine, (Fig. 1). A much smaller induction of ODC was seen in cerebral cortex
"* 800
p< 0.05 p< 0.005
7
c::
>
T
E O
T
600
(,9 400
o
I
I 200
,-,---IT
*
qulnacrine 0 100 200 mg/kg
indomethacin 0 30 60 rag/ kg
NDGA 0
30
mg/kg
Fig. 1.
Effect o f quinacrine, indomethacin and NDGA on induction of ODC in cerebral cortex by unilateral injection of kainate. A unilateral injection of kainate was made into the nucleus basalis and cerebral cortex was removed at 8 hours from the ipsilateral and contralateral hemispheres. Animals were treated with either vehicle or drug at the time of lesion. Vehicles used were water for quinacrine, and 1 M NazCO3 for indomethacin and NDGA. Values are means (picomoles 14CO2 released/rag protein/h) with the SEMs shown by error bars. Values in the contralateral cortex are shown as hatched bars superimposed on clear histograms for the ipsilateral cerebral cortex. The number of animals in each group was 6-10. The asterisks indicate the level of significance when comparing drug treated groups with vehicle treated groups. Students' t test (tmpaired). contralateral to the kainate injection but this was only inhibited by quinacrine at 200 mg/kg. Treatment with dexamethasone at the time of lesion produced a dose dependent attenuation in the response, which was significant at doses of 1 mg/kg and above. The greatest attenuation in response was seen at a dose of 2 mg/kg where the response was reduced by 67% in dexamethasone-treated animals compared to vehicle treated animals (Fig. 2). The induction of ODC in contralateral cortex was also significantly reduced at this dose of dexamethasone but not at other doses. Effects of cyclo-oxygenase and lipoxygenase inhibitors on the induction of ODC. The involvement of prostaglandins or leukotrienes in mediating the induction of ODC was investigated by the use of indomethacin and NDGA. Administration of indomethacin at the time of lesion caused a significant attenuation of the induction of ODC in cerebral cortex ipsilateral to the kainate injection. This effect was significant at a dose of 30 mg/kg where an inhibition of 38% was obtained. The induction o f ODC was further attenuated (58%) at 60 mg/kg (Fig.l). The lipoxygenase inhibitor NDGA was without effect on the induction of ODC up to a dose of 30 mg/kg. It was not possible to use a higher dose of this agent due to its poor solubility.
PL-80
Excitotoxin
700 ._Cl (1)
and O D C in C e r e b r a l
Cortex
Vol.
51, No.
8, 1992
o-- c o n t r a l a t e r a l ipsilateral
~
600
500
o
O_
O
O E3% O E
400
Ob
O_
300
200
v
100 0
I
0
. . . . . . . .
I
. . . . . . . .
.01
I
. . . . . . . .
.1
I
1
. . . . . . . .
I
10
dexamethasone (mg/kg) Fig. 2. Effect of dcxan/elhasone on the induction of ODC by kainate. Animals were treated with vehicle (0) or dexamethasone at a range of concentrations from 0.01 to 3 mg/kg. ODC was measured in ipsilateral or contralateral cereb,'al cortex at 8 hours as described in the Legend to Fig. 1. Values are expressed as Fig. 1. The number of animals used was 6-10 lit each concentration of the drug. The asterisk and double asterisk indicates that the wflues all significantly reduced when compared to the vehicle injected group with a significance of p
