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Turnover of monoamines in hippocampus of rats fed on vitamin E-deficient diet
Brain Research, 604 (1993) 154-159 " © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
154
BRES 18539
Turnover of monoamines in hippocampus of rats fed on vitamin E-deficient diet Ang61ica Castafio *, Maria Luisa Vizuete * *, Josefina Cano and Alberto Machado Departamento de Bioqu[mica, Bromatolog{a y Toxicolog{a, Facultad de Farmacia, Unit~ersidadde Sevilla, Set;illa (Spain)
(Accepted 15 September 1992)
Key words: Vitamin E; Hippocampus; Monoamine; Adult; Rat
Turnover of noradrenaline (NA), dopamine (DA), serotonin (5-hydroxytryptamine; 5-HT) and their metabolites has been measured after a 15-day vitamin E-deficient diet in adult hippocampus. Moreover, we have measured in vitro receptor binding of [3H]5-HT in hippocampal membranes from control and vitamin E-deficient rats. Turnover rates of 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxy-3-indolacetic acid (5-HIAA) have been assayed from the disappearance rates after blocking by pargyline inhibition of monoamine oxidase (MAO). DA, NA, 5-HT, normetanephrine (NMN) and 3-methoxytyramine (3-MT) turnover rates have been measured as accumulation rates of DA, NA, 5-HT, NMN and 3-MT after pargyline inhibition of MAO. An increase was found in the turnover rate of DA between control and experimental animals. In contrast, no changes were found in the turnover rate of 3-MT and DOPAC. No change was found in the turnover rate of NA although there was an increase of the turnover rate of NMN in vitamin E-deficient diets. No change was found in the turnover rate of 5-HT although there was a decrease of 5-HIAA turnover rate in the animals fed on a low vitamin-E diet. With respect to the 5-HT 1 receptors, no changes were found in the affinity (k~) but the receptor number (Bmax) was increased in vitamin E-deficient rats.
INTRODUCTION T h e h i p p o c a m p u s is k n o w n to b e a c e n t r a l c o m p o n e n t of m e m o r y - r e l a t e d n e u r a l systems. I n p a r t i c u l a r , t h e p y r a m i d a l n e u r o n s of the r e g i o n s u p e r i o r have b e e n shown to b e n e c e s s a r y for this p r o c e s s 36. Dysfunctions of t h e h i p p o c a m p u s are involved in the m e m o r y deficits a s s o c i a t e d with A l z h e i m e r ' s d i s e a s e 38. It is known that axons c o n t a i n i n g d o p a m i n e ( D A ) , n o r a d r e n a l i n e ( N A ) a n d s e r o t o n i n ( 5 - H T ) c o n v e r g e to t h e h i p p o c a m p u s . T h e r e is e v i d e n c e to s u p p o r t a D A p r o j e c t i o n to h i p p o c a m p u s f r o m V T A or s u b s t a n t i a n i g r a 3. T h e h i p p o c a m p a l f o r m a t i o n has s u b s t a n t i a l p r o j e c t i o n s of N A - c o n t a i n i n g fibres a n d 5 - H T fibres from t h e locus c e r u l e u s 21 a n d r a p h e nuclei, respectively 2'2°. It has b e e n r e p o r t e d that D A d e c r e a s e d t h e s p o n t a n e o u s activity o f h i p p o c a m p a l p y r a m i d a l cells o r d e p r e s s e d g l u t a m a t e - i n d u c e d firing 3. B o t h N A a n d 5 - H T t e n d to s u p p r e s s s p o n t a n e o u s or g l u t a m a t e - i n d u c e d firing of
p y r a m i d a l cells a n d g r a n u l e s cells in t h e h i p p o c a m pus 34'19. A significant b o d y o f e v i d e n c e suggests t h a t s e r o t o n i n ( 5 - H T ) m a y play an i m p o r t a n t role in t h e m e d i a t i o n of t h e p r o c e s s e s u n d e r l y i n g l e a r n i n g a n d m e m o r y 28,29. 5 - H T a p p e a r s to inhibit t h e firing of c h o l i n e r g i c n e u r o n s within t h e h i p p o c a m p u s 23'31. A b n o r m a l f u n c t i o n i n g of t h e s e r o t o n i n e r g i c system has b e e n i m p l i c a t e d in d e m e n t i a o f t h e A l z h e i m e r type 8'9. P a t h o l o g i c a l c h a n g e s in t h e s e r o t o n i n e r g i c r a p h e syst e m m a y i m p a i r m e m o r y 41. It is well e s t a b l i s h e d t h a t v i t a m i n E scavenges t h e p e r o x y r a d i c a l in lipid p e r o x i d a t i o n 11. In r e c e n t y e a r s it has b e c o m e clear t h a t v i t a m i n E is i m p o r t a n t for norm a l n e u r o l o g i c a l f u n c t i o n 26'18. O x y g e n - d e r i v e d free r a d i c a l s a r e highly reactive c h e m i c a l species which, as well, have b e e n i m p l i c a t e d in t h e n e u r o l o g i c a l s e q u e l a e a s s o c i a t e d with v i t a m i n - E deficiency 16. F r e e radicals a l o n e m a y i n d u c e cell d e a t h by d a m a g i n g lipids or p r o t e i n s , while r e a c t i o n s b e t w e e n free radicals a n d
Correspondence: A. Machado, Departamento de BioqMmica, Bromatologla y Toxicologla, Facultad de Farmacia, Universidad de Sevilla, c/o Prof.'Garcla Gonz~lez, s/n, 41012 Sevilla, Spain. Fax: (34) (5) 423 37 65. * Recipient of a predoctoral fellowship from the Junta de Andalucla. ** Recipient of a postdoctoral fellowship from the Junta de Andalucia.
155 neurotransmitters may lead to formation of endogenous neurotoxin 4°. It has been reported that indolamines and catecholamines act as free radical scavengers 39. Interaction of neurotransmitters and free radicals may result in formation of endogenous neurotoxins. A serotoninergic neurotoxin, 5,7-dihydroxytryptamine, was reported to be formed by free radical oxidation of 5-HT in vitro 44. Moreover, it has been reported that 5-HT is oxidized by the Fenton reaction or by hypoxantine-xantine oxidase-mediated reaction in vitro, it is transformed into tryptamine-4,5-dione (4,5-DKT) 39. Little is known about the action of free radical scavengers on the neurotransmitters. The purpose of this study was to examine what effect a-tocopherol deficiency might have on the turnover of the monoamines and 5-HT 1 receptor in the rat hippocampus. We have used non-isotopic methods to estimate the turnover rate of DA, N A and 5-HT.
solvent mixtures were filtered by vacuum through a 0.45 /xm filter, and degassed by vacuum prior to ultrasound bath in order to eliminate air bubbles, which interfere with the electrochemical assay. Stock standards were prepared by accurately weighing 4 mg of each of the standard and dissolving in 4 ml 0.1 M perchloric acid and 1 m M sodium bisulfite. The stock was then stored at + 4 ° C for 2 months. The brain tissue was homogenized in 0.1 M perchloric acid containing 1 m M sodium bisulfite by ultrasonic disintegration over ice using a Labsonic 1510. Samples were centrifuged at 30,000 g for 15 min at + 4°C and the supernatant was then filtered through a 0.2 /xm filter. Material was injected by high-pressure injection valve (Rheodine), with a 20 /xl sample loop. Analyses were performed in the isocrafic mode, at a flow rate of 0.8 m l / m i n and at room temperature. Concentrations in brain samples were calculated with the aid of (linear) calibration curves obtained after the injection of pure standard. To identify small amounts in chromatograms when compounds are found near the detection limit, there are, in general, several possible ways to confirm the identity of these compounds. We carried out: Chromatograms of both a purified brain extract and a standard solution recorded at various oxidation potentials. The authentic and studied compounds should have similar electrochemical behaviour. The electrochemical behaviour of endogenous, e.g., D A and 3-MT, isolated from the brain tissue, were compared with corresponding standard solutions. The similarity of the voltagrams supports the specificity of the assay.
MATERIALS AND METHODS
NA, NMN, DA, 3-MT and 5-HT accumulation after MAO inhibition.
Animals, treatment and dissection . A group of 60 female rats of the Wistar strain weighing 200-250 g were used: 36 for m e a s u r e m e n t of turnover and 24 for binding experiments. Rats were fed semisynthetic diets containing the following vitamin E concentrations: (i) Basal diet, 170 m g / k g (Panlab S.L.); (ii) Vitamin E-deficient diet, less than 15 m g / k g (Panlab S.L.). Animals were housed two per cage, with a 12 h l i g h t / d a r k cycle, and with free access to the diets and to water. The animals were regularly weighed and their food consumption recorded. After 15 days of being fed the vitamin E-deficient diet, they were sacrificed by decapitation between 10.00 h and 11.00 h and the brains quickly removed. A cut was m a d e through the corpus callosum and the cortex was peeled back exposing the hippocampus which was carefully removed and frozen in liquid N 2 until analysis. The total time for the isolation of the tissues was less than 3 min. Pargyline HCI (75 m g / k g ) , was dissolved in 0.9% NaCI and injected intraperitoneally. Injection volume was 5 m l / k g . Products. [3H]5-hydroxytryptamine creatine sulphate (23.4 C i / m m o l ) was purchased from New England Nuclear. Pargyline HCL, 5-HT, 5HIAA, NA, NMN, D A and 3-MT were purchased from Sigma (St. Louis, MO).
Measurement of turnover Assay procedure. Analysis was performed by m e a n s of H P L C equipped with a Kontron 420 p u m p in conjunction with electrochemical detector (Bioanalytical Systems, LC-4B). Measurement of biogenic amines and their metabolites in brain. The mobile phase was prepared according to DiBussolo et al. 1°. The
NA, NMN, DA, 3-MT and 5-HT levels were determined at various times (0-20 min) after intraperitoneal injection of pargyline. NA, NMN, DA, 3-MT and 5-HT accumulation rates were obtained by calculating the slope (b) of the curve by linear regression. In this case, the standard deviation of each accumulation rate equalled standard deviation of b (SDh). Slopes of the regression lines of control and vitamin E-deficient animals were compared using the Student's t-test. Turnover of acid metabolites; DOPAC, HVA and 5-HIAA. Concentrations of D O P A C , H V A and 5 - H I A A were determined at various times (0-20 rain) after intraperitoneal injection of pargyline (75 m g / k g ) . From a semilog plot of D O P A C , H V A and 5 - H I A A concentrations versus time the slope of decline was obtained using linear regression. The slopes (b) of the regression lines were calculated by the m e t h o d of least squares. The slopes obtained were used to estimate total D O P A C , H V A and 5 - H I A A turnover rate (T) as the product of fractional rate constant (k) k = b / 0 . 4 3 4 and the estimated concentration of each metabolite at time 0 (steady state) 5. Standard deviation of k (SDk) equalled standard deviation for b (SD b) divided by 0.434. Slopes of the regression lines and the corresponding turnover rates of control and vitamin E-deficient animals were compared using the Student's t-test. 5-HT1 receptor binding assay. 5-HT 1 sites were measured by a modification of the procedure of Gozlan et al. 15. Saturation binding analyses were conducted with 9 concentrations of [3H]5-HT (0.5-15 nM) with non-specific binding estimated by competition with 10 p.M of 'cold' 5-HT. M e m b r a n e s were prepared from frozen tissue by homogenization (10 s Polytron, setting 5) in 10 vol of 50 m M Tris-HC1 buffer, pH 7.4
TABLE I
Turnover of DA, 3-MT, DOPAC, NA and NMN in hippocampus of controls and vitamin E-deficient rats after treatment with pargyline Results are given as mean_+ SD of 6 determinations. Statistical significance: * P < 0.01 low vitamin E-deficient rats vs. controls (Student's t-test).
Metabolite DA 3-MT DOPAC NA NMN
Steady state (pmol / g)
Turnover (pmol. g. h 1)
K (h 1)
Control
Low vit. E
Control
Low vit. E
Control
Low vit E
106.8_+ 31.9 62.2_+ 12.7 85.3+ 13.9 1 231.4 -+ 185.1 77.5-+ 6.8
180.4_+ 19.5 * 56.4+_ 30.4 73.1-+ 8.4 904.8 _+116.8 * 144.3_+ 35.5 *
2.7+_0.3 -
2.5_+0.3 -
469.7-+ 60.4 103.9_+ 20.7 230.3_+ 62.6 686.3 -+ 299.3 299.4_+ 29.9
1038.0_+ 166.1 * 122.9_+ 60.3 184.2_+ 17.4 641.5 _+210.6 486.5_+ 95.9 *
156 T A B L E Il
TurnoL'er of 5-HT and 5-HIAA in hippocampus of controls" and uitamin E-deficient rats after treatment with pargyline
Results are given as mean_+SD of six determinations. Statistical significance: * P < 0.05; * * P < 0.01 low vitamin E-deficient rats vs. controls (Student's t-test). Metabolite
Steady state (pmol / g)
5-HT 5-HIAA
Turnot;er (pmol. g" h - 1)
K (h - 1)
Control
Low L,it. E
Control
Low uit. E
Control
Low uit. E
1500.3 + 167.9 1319.2 _+196.5
1042.8 _+120.8 * * 843.2 + 112.2 * *
1.4 + 0.2
1.1 _+0.2 *
3 228.4 + 286.0 1846.9 +_552.2
3 428.1 _+346.9 885.4 + 269.5 * *
containing 4 nM CaC12, 1 mM EDTA and 0.1 mM PMSF. Membranes were pelleted by centrifugation (15 rain at 40,000 g). The resulting pellet was washed twice more before resuspension in 10 vols. of 50 mM Tris-HCl, pH 7.4, containing 4 mM CaCI2, 1 mM EDTA, 10 IzM pargyline and 0.1% ascorbic acid. The assay was initiated by addition of 50 /~1 of the membrane suspension (equivalent to 0.25-0.3 mg prot) to tubes containing [3H]5-HT and proceeded for 30 rain at 23°C. Following incubation, the assay mixture were rapidly filters under vacuum through glass microfiber filters (GF/B) with two 5-ml washes using 50 mM Tris-HCl buffer (pH 7.4). Dried filters were counted by liquid scintillation spectroscopy. Data analysis. The Scatchard transformation of the saturation curves were adjusted using the program LIGAND 27. The comparison between the means of different groups of data was evaluated by a Student's t-test. When the P-value was smaller than 0.05 (*) or smaller than 0.01 (**), the difference was considered significant.
inhibition by pargyline. T h e fractional rate c o n s t a n t an d the c o r r e s p o n d i n g t u r n o v e r did n o t c h a n g e bet w e e n b o t h g r o u p s of animals.
Accumulation rate o f 5-HT and turnover o f 5-HIAA. T h e 5 - H T and 5 - H I A A steady-state levels w e r e l o w e r in v i t a m i n E - d e f i c i e n t rats t h a n in controls (30.5% and 36.1%, respectively, P < 0.01; T a b l e II). A f t e r pargyline t r e a t m e n t 5 - H T s h o w e d in controls a l i n e a r accumulation
and
5-HIAA
an
exponential
declination
t h r o u g h the t i m e intervals st u d i ed (Figs. 2 A a nd 2B, respectively).
80C A
RESULTS
Body weight gain and food intake.
Body w e i g h t gain
B E Q.
400
. / ~
and food intake did not vary significantly b e t w e e n t h e control an d v i t a m i n E - d e f i c i e n t animals, suggesting that
2o0
the deficiency of v it a m i n E for 15 days was n o t detri-
o
m e n t a l to their growth. F o o d intake: controls, 16.1 _+ 2.7
3000
weight
250C
controls,
30.0_+ 4.8
g . (animal)
days)-~; v i t am i n E - d e f i c i e n t animals, ( a n i m a l s ) - 1. (15 d a y s ) - 1.
g.
Turnover o f monoamines Accumulation rate of DA, 3-MT, NA, N M N and turnover of DOPAC. A f t e r pargyline t r e a t m e n t , D A , 3-MT, N A
6
2'0
B
1 .(15
33.4_+7.8
~ rain
g d i e t . (animal) - 1 . (day)-~; v i ta m in E - d e f i c i e n t animals, 16.8 _+ 0.3 g diet • ( a n i m a l ) - 1 . (day)-~. Body gain:
~
~
200C
,< x
100C 5OO 00
' 10
2'0 rain
and N M N showed a linear a c c u m u l a t i o n t h r o u g h the 50C
t i m e intervals s t u d ie d in b o t h groups of animals (Figs.
C
1A for D A , 1B for N A and 1C for N M N ) . T h e D A
40C
steady-state c o n c e n t r a t i o n o f v i t a m i n E - d e f i c i e n t rats E
i n c r e a s e d with r e s p e c t to t h e controls (69.0%, P < 0,01)
o_
along with the D A a c c u m u l a t i o n rate (120.9%, P <
z
0.01; T a b l e I).
z
300
2OO 100 "
N o significant d i f f e r e n c e s w e r e f o u n d in the accum u l a t i o n rates of 3 - M T and N A b e t w e e n b o t h groups of animals (Table I). O n the contrary, v i t a m i n E- d ef i cient rats s h o w e d h i g h e r a c c u m u l a t i o n rate of N M N t h a n the controls (62.5%, P < 0.01; T a b l e I). DOPAC
levels d e c l i n e d e x p o n e n t i a l l y after M A O
00
i
1~3
20
_
rain
Fig. 1. Concentration of DA (A), NA (B) and NMN (C) in adult rat hippocampus of controls (open circles) and vitamin E-deficient animals (filled circles) at various times after MAO inhibition by pargyline (75 mg/kg). Each point is the mean _+S.D. of six determinations.
157 5-HT1 receptor binding. Fig. 3 shows the Scatchard analysis of [3H]5-HT binding to control and deficient vitamin E rat hippocampal membranes. The Scatchard was linear showing a single high-affinity in both groups of animals ( K a = 2.97 _+ 0.57 nM for controls and 3.41 _+ 1.17 nM for vitamin E-deficient rats). However the Bma x of vitamin E-deficient rats was higher (25.5%, P < 0.01) than in controls. Bmax w e r e : 126.7_+ 12.2 f m o l / m g prot in control rats and 159.0 _+ 11.5 f m o l / m g prot in vitamin E-deficient rats.
3000 A 250C 03 E 200C Q. I-]:
150C
t
I00C OI
o
~b
2'o rain
DISCUSSION
E .< .<
1000 800
to
d o
~b
23 rain
Fig. 2. Concentration of 5-HT (A) and semilogarithmic plot of concentration of 5-HIAA (B) in adult rat hippocampus of controls (open circles) and vitamin E-deficient animals (filled circles) at various times after MAO inhibition by pargyline (75 mg/kg). Each point is the mean _+S.D. of six determinations.
No significant changes were found in the accumulation rate of 5-HT between both groups of animals (Table II). With respect to 5-HIAA, its fractional rate constant ( K ) decreased (21.4%; P < 0.05) along with the resulting turnover (52.1%; P < 0.01) comparing deficient vitamin E with control values (Table II) 5OO 400 . ~ , , , ~ e •
LL
o controt ~
• vit, E
rn 30O
2OO
IOO
3(
50 (3H)-5HT
1OO
150
bound (fmol/mg prot)
Fig. 3. Scatchard analysis of [3H]-3HT binding activity in control (open circles) and vitamin E-deficient (filled circles) hippoeampal
membranes. Results are representative of six experiment done in duplicate. Lines were drawn by linear regression using the parametres (dissociation constant and Bmax) calculated by the program LIGAND.
Vitamin E-deficient rats showed a significant increase in hippocampus DA accumulation rate compared with control animals. On the contrary, NA and 5-HT accumulation rates were unchanged but there was a significant decrease of 5-HT and NA levels. There are several reasons to assume that all these changes were induced by the vitamin-E deficiency: (i) both diets, standard and vitamin E-deficient, were supplied by the same manufacturer, ensuring identical diet composition except in vitamin-E content; (ii) there were no differences in the food intake and corporal weight gain between groups, showing identical palatability in both diets; and (iii) it has been reported previously that similar levels of vitamin-E deficiency to that studied by us induced a significant decrease in vitamin-E content in plasma 39. Although the amount of D A in the hippocampus of the rat is only 10% of hippocampal NA, direct assessment of the DA turnover rate, by measuring DA accumulation after monoamine oxidase inhibition or the rate of D O P A C disappearance, indicated that D A is more quickly metabolized in the hippocampus than in other brain areas, including striatum 42. Therefore, due to this fast turnover rate in mesohippocampal DA, the hippocampus could be affected by free radicals in a different way to other brain structures. Some observers believe that the breakdown products of DA, which include peroxides that eventually generate cytotoxic free radicals, might in fact be the cause of tissue degeneration 3°. If this is so, then the increased DA turnover as consequence of vitamin-E deficiency would increase the risk of cell damage. In spite of an evident lack of enough data, it appears that D A can influence the excitability of hippocampal cells and thus may function as a neurotransmitter or a neuromodulator 3. After vitamin-E deficiency the NA accumulation rate was unchanged, but the NMN turnover was increased with respect to the controls. This increase in catabolism would be the cause of the decrease of NA levels after
158 vitamin-E deficiency. It has been reported that N A modulates the release of glutamate from dentate but not from C A J C A 3 of the hippocampus 22. Therefore, the above paper and another 37 have suggested that this structure may possess greater regional heterogeneity with respect to synaptic plasticity and neuromodulation than has previously been appreciated. It is interesting that vitamin-E deficiency during a short period produced a significant decrease in the 5-HT levels (30.5%). However, even though there was no significant difference in the 5-HT accumulation rate, the 5 - H I A A turnover decreased significantly. One possible way to explain these results would be that, since the 5-HT synthesis does not change and the 5-HT levels decrease, the serotoninergic fibers could have either increased its release or decreased its uptake capacity. Consequently, there was an increase of 5-HT in the synaptic cleft. As the deficit of vitamin E produces an increase of free radicals, part of this free 5-HT may be oxidized into a neurotoxic substance, such as tryptamine-4,5-dione (4,5-DKT) with a consequent decrease of catabolism. The increase of 5-HT release could be due to the action of the oxidative products. It has been reported that 4,5-DKT increases the release of 5-HT without affecting the release of D A 6. Recent experiments have provided evidence supporting the hypothesis that extracellular accumulation of synaptically released neurotransmitters, such as serotonin, have been thought to participate in the development of delayed neuronal death ~3, which could be due to the products of oxidation of 5-HT. When the 4,5-DKT, a partially oxidized form of serotonin, was injected directly into the hippocampus it produced axon terminal degeneration in sectors CA 1 and C A 3 and in the dentate gyrus 7. Moreover, it has been reported that partially oxidized serotonin (tryptamine4,5-dione) exhibits a striking propensity for medial limbic system structures especially hippocampal, entorhinal and cingulate cortices 7. To go deeper into the effect of vitamin E on 5-HT, we have studied 5-HT 1 receptors, since within the hippocampus they are the major class of serotonin receptor, and have high affinity for serotonin and its agonists 4'25. Moreover, in post-synaptic neurons, the levels of most neurotransmitter receptors are regulated in response to transynaptic activity. Receptor supersensitization (upregulation) is an adaptive response of the post-synaptic neuron to the functional loss of pre-synaptic input. Our results show that the deficit of vitamin E increased the number of 5-HT 1 binding sites in the hippocampus without affecting receptor affinity. It has been reported that serotonin receptors (5-HTa and
5-HT 2) are more resistant to modulation than their dopaminergic counterparts and that the mechanism for their modulation may be more complex for serotoninergic systems z4. Denervation lesions of serotoninergic input with 5,7-dihydroxytryptamine do not increase 5H T z binding sites 35'43. Only drugs which primarily increase serotonin concentration in the synaptic cleft are effective in down-regulation 5-HT 1 receptor binding sites 32,33A4,12. Therefore, reduced levels of serotonin could result in an up-regulation of 5-HT 1 receptors. Alternatively, there could be alterations in serotoninergic synapses which produce the increase in 5-HT1 receptors. Several aspects of the changes in 5-HT 1 receptors as a result of vitamin-E deficiency provide interesting correlations with the important modulatory role ascribed to the 5-HT in hippocampus. There is evidence to support of an important neuromodulatory role for 5-HT in the mediation of learning and memory within the hippocampus 1. This data suggests that a deficiency of vitamin E during short periods leads to significant changes of D A and 5-HT metabolism. Recently, it has been reported that vitamin E prevented CA 1 pyramidal cell death following ischemia in the gerbil 17. An understanding of the mechanism of serotoninergic neurotoxicity would allow possible high-risk brain structures to be evaluated and protective therapeutic treatments to be assessed. This work was supported by a grant from the Fundaci6n Ram6n Areces.
Acknowledgements.
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4 Blurton, P.A. and Wood, M.D., Identification of multiple binding sites for [3H]5-hydroxytryptaminein the rat CNS, J. Neurochem., 46 (1986) 1392-1398. 5 Brodie, B.B., Costa, E., Dlabac, A., Neff, N.H. and Smookler, H.H., Application of steady-state kinetics to the estimation of synthesis rate and turnover time of tissue catecholamines, J. Pharmacol. Exp. Ther, 55 (1966) 493-498. 6 Chen, J.C., Crino, P.B., Schnepper, P.W., To, A.C.S. and Volicer, L., Increased serotonin effiux by a partially oxidized serotonin: tryptamine-4,5-dione, J. Pharmacol. Exp. Ther., 250 (1989) 141148. 7 Crino, P.B., Vogt, B.A., Chert, J.C. and Volicer, L., Neurotoxic effects of partially oxidized serotonin: tryptamine-4,5-dione,Brain Res., 504 (1989) 247-257.
159 8 Cross, A.J., Cross, T.J., Johnson, J.A., Perry, E.K., Perry, R.H., Blessed, G. and Tomlinson, B.E., Studies on neurotransmitter receptor systems in neocortex and hippocampus in senile dementia of the Alzheimer type, J. Neurol. Sci., 64 (1984) 109-117. 9 Curcio, C.A. and Kemper, T., Nucleus raphe dorsalis in dementia of the Alzheimer type: neurofibrillary changes and neuronal packing density, Z Neuropathol. Exp. Neurol., 43 (1984) 359-368. 10 DiBussolo, J.M., Gant, J.R. and Kerber, J.D., Instrumental considerations in catecholamine analysis using liquid chromatography with electrochemical detection, Chromatog. Newslett., 11 (1983) 27-29 11 Doba, T., Burton, G.W. and Ingold, K.U., Antioxidant and co-antioxidant activity of vitamin C. The effect of vitamin C, either alone or in the presence of vitamin E or a water-soluble vitamin E analogue, upon the peroxidation of aqueous multilamellar phospholipid liposomes, Biochim. Biophys. Acta, 835 (1985) 298303. 12 Dumbrille-Ross, A. and Tang, S.W., Manipulations of synaptic serotonin: discrepancy of effects on serotonin S 1 and S 2 sites, Life Sci., 32 (1983) 2677-2684. 13 Fujikura, H., Kato, H., Nakano, S. and Kogure, K., A serotonin $2 antagonist, naftidrofuryl, exhibited a protective effect on ischemic neuronal damage in the gerbil, Brain Res., 494 (1989) 387-390. 14 Fuxe, K., Ogren, S.O., Agnati, L.F., Eneroth, P., Holm, A.C. and Anderson, A., Long-term treatment with zimelidine leads to a reduction in 5-hydroxytryptamine neurotransmission within the central nervous system of the mouse and rat, Neurosci. Lett., 21 (1981) 57-62. 15 Gozlan, H., Emerit, M.B., Hall, M.D., Nielsen, M. and Hamon, M., In situ molecular sizes of the various types of 5-HT binding sites in the rat rabbit rat, Biochem. Pharmacol., 35 (1986) 18911897. 16 Halliwell, B. and Gutteridge, J.M.C., Oxygen radicals and the nervous system, Trends Neurosci., 8 (1985) 22-26. 17 Hara, H., Kato, H. and Kogure, J., Protective effect of o~tocopherol on ischemic neuronal damage in the gerbil hippocampus, Brain Res., 510 (1990) 335-338. 18 Harding, A.E., Vitamin E and the nervous system, Crit. Rev. NeuroL, 3 (1987) 89-103. 19 Jahnsen, H., The action of 5-hydroxytryptamine on neuronal membranes and synaptic transmission in area CA 1 of the hippocampus in vitro, Brain Res., 197 (1980) 83-94. 20 K6hler, C., On the serotonergic innervation of the hippocampal region: an analysis employing immunohistochemitry and retrograde fluorescent tracing in the rat brain. In S.L. Palay and V. Chan-Palay (Eds.), Cytochemical Methods in Neuroanatomy, Alan R. Liss, New York, 1982, pp. 387-405. 21 Loy, R., Koziell, D.A., Lindsey, J.D. and Moore, R.Y., Noradrenergic innervation of the adult rat hippocampal formation, J. Comp. Neurol., 189 (1980) 699-710. 22 Lynch, M.A. and Bliss, T.V.P., Noradrenaline modulates the release of (14C)glutamate from dentate but not from CA 1 / C A 3 slices of the rat hippocampus, Neuropharmacology, 25 (1986) 493-498. 23 Maura, G. and Raiteri, M., Cholinergic terminals in rat hippocampus possess 5-HT m receptors mediating inhibition of acetylcholine release, Eur. J. Pharmacol., 129 (1986) 333-337. 24 May, P.C., Morgan, D.G. and Finch, C.E., Regional serotonin receptor studies: chronic methysergide treatment induces a selective and dose-dependent decrease in serotonin-2 receptors in mouse cortex, Life Sci., 38 (1986) 1741-1747. 25 Mower, G.D., Comparison of serotonin 5-HT1 receptors and innervation in the visual cortex of normal and dark-reared cats, J. Comp. Neurol., 312 (1991) 223-230.
26 Muller, D.P.R., Lloyd, J.K. and Wolff, O.H., Vitamin E and neurological function, Lancet, i (1983) 225-228. 27 Munson, P.J. and Rodbard, D., LIGAND: a versatiIe computerized approach for characterization of ligand-binding system, Anal Biochem., 107 (1980) 220-239. 28 Normile, H.J. and Altman, H.J., Serotonin, Alzheimer's disease and learning and memory in animals. In H.J. Altman (Ed.), Alzheimer's Disease: Problems, Prospects and Perspectives, Plenum, New York, 1988, pp. 141-156. 29 Ogren, S.O., Central serotonin neurons and learning in the rat. In N.N. Osborne (Ed.), Biology of Serotonergic Neurotransmission, Oxford University Press, Oxford, 1982, pp. 317-334. 30 Pellmar, T.C., Neel, K.L. and Lee, K.H., Free radicals mediate peroxidative damage in guinea pig hippocampus in vitro, J. Neurosci. Res., 24 (1989) 437-444. 31 Robinson, S.E., Effect of specific serotonergic lesions on cholinergic neurons in the hippocampus, cortex and striatum, Life Sci., 32 (1983) 345-353. 32 Savage, D.D., Mendels, J. and Frazer, A., Monoamine oxidase inhibitors and serotonin uptake inhibitors: differential effects of [3H]serotonin binding sites in rat brain, J. Pharmacol. Exp. Ther., 212 (1980a) 259-263. 33 Savage, D.D., Mendels, J. and Frazer, A.,Decrease in [3H]serotonin binding in rat brain produced by the repeated administration of either monoamines oxidase inhibitors of centrally acting serotonin agonists, Neuropharmacology, 19 (1980b) 1063-1070. 34 Segal, M. and Bloom, F.E., The action of norepinephrine in the rat hippocampus. I. Iontophoretic studies, Brain Res., 72 (1974) 79-97. 35 Segawa, T., Mizyuta. and Nomura, Y., Modifications of central 5-hydroxytriptamine binding sites in synaptic membranes from rat brain after long-term administration of tricyclic antidepressents, Eur. J. Pharmacol., 58 (1979) 75-83. 36 Squire, L.R., Mechanisms of memory, Science, 232 (1986) 16121619. 37 Stanton, P.K. and Sarvey, J.M., Depletion of norepinephrine, but not serotonin, reduces long-term potentiation in the dentate gyrus of rat hippocampal slices, J. Neurosci., 5 (1985) 2169-2176 38 Van Hoesen, G.W., Hyman, B.T., Damasio, A.R., Cell-specific pathology in neural systems of the temporal lobe in Alzheimer's disease. In Swabb, D.F., Fliers, E., Mirmiran, M., Van Gool, W.A. and Van Haaren, F. (Eds), Progress in Brain Research, Elsevier, Amsterdam, 1986, pp. 321-335. 39 Vatassery, G.T., Angerhofer, C.K. and Peterson, F.J., Vitamin E concentrations in the brains and some selected peripheral tissues of selenium-deficient and vitamin E-deficient mice, J. Neurochem., 42 (1984) 554-558. 40 Volicer, L. and Crino, P.B., Involvement of free radicals i n dementia of the Alzheimer type: a hypothesis, Neurol. Aging, 1.t (1990) 567-571. 41 Wenk, G., Hughey, D., Boundy, V. and Kim, A., Neurotransmitters and memory: role of cholinergic, serotonergic, and noradrenergic systems, Behav. Neurosci., 101 (1987) 325-332. 42 Westerink, B.H.C., Bosker, F.J. and Wirix, E., Formation and metabolism in nine areas of the rat brain: 'modifications by haloperidol, J. Neurochem., 42 (1984) 1321-1327. 43 Whitaker, P.M. and Deakin, J.F.W., Does [3H]serotonin label presynaptic receptors in rat frontal cortex?, Eur. Z Pharmacol., 73 (1981) 349-351. 44 Wrona, M.Z., Lemordant, D., Lin, L., Blank, C.L. and Dryhurst, G., Oxidation of 5-hydroxytryptamine and 5,7-dihydroxytryptamine. A new oxidation pathway and formation of a novel neurotoxin, Z Med. Chem., 29 (1986) 499-505.
154
BRES 18539
Turnover of monoamines in hippocampus of rats fed on vitamin E-deficient diet Ang61ica Castafio *, Maria Luisa Vizuete * *, Josefina Cano and Alberto Machado Departamento de Bioqu[mica, Bromatolog{a y Toxicolog{a, Facultad de Farmacia, Unit~ersidadde Sevilla, Set;illa (Spain)
(Accepted 15 September 1992)
Key words: Vitamin E; Hippocampus; Monoamine; Adult; Rat
Turnover of noradrenaline (NA), dopamine (DA), serotonin (5-hydroxytryptamine; 5-HT) and their metabolites has been measured after a 15-day vitamin E-deficient diet in adult hippocampus. Moreover, we have measured in vitro receptor binding of [3H]5-HT in hippocampal membranes from control and vitamin E-deficient rats. Turnover rates of 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxy-3-indolacetic acid (5-HIAA) have been assayed from the disappearance rates after blocking by pargyline inhibition of monoamine oxidase (MAO). DA, NA, 5-HT, normetanephrine (NMN) and 3-methoxytyramine (3-MT) turnover rates have been measured as accumulation rates of DA, NA, 5-HT, NMN and 3-MT after pargyline inhibition of MAO. An increase was found in the turnover rate of DA between control and experimental animals. In contrast, no changes were found in the turnover rate of 3-MT and DOPAC. No change was found in the turnover rate of NA although there was an increase of the turnover rate of NMN in vitamin E-deficient diets. No change was found in the turnover rate of 5-HT although there was a decrease of 5-HIAA turnover rate in the animals fed on a low vitamin-E diet. With respect to the 5-HT 1 receptors, no changes were found in the affinity (k~) but the receptor number (Bmax) was increased in vitamin E-deficient rats.
INTRODUCTION T h e h i p p o c a m p u s is k n o w n to b e a c e n t r a l c o m p o n e n t of m e m o r y - r e l a t e d n e u r a l systems. I n p a r t i c u l a r , t h e p y r a m i d a l n e u r o n s of the r e g i o n s u p e r i o r have b e e n shown to b e n e c e s s a r y for this p r o c e s s 36. Dysfunctions of t h e h i p p o c a m p u s are involved in the m e m o r y deficits a s s o c i a t e d with A l z h e i m e r ' s d i s e a s e 38. It is known that axons c o n t a i n i n g d o p a m i n e ( D A ) , n o r a d r e n a l i n e ( N A ) a n d s e r o t o n i n ( 5 - H T ) c o n v e r g e to t h e h i p p o c a m p u s . T h e r e is e v i d e n c e to s u p p o r t a D A p r o j e c t i o n to h i p p o c a m p u s f r o m V T A or s u b s t a n t i a n i g r a 3. T h e h i p p o c a m p a l f o r m a t i o n has s u b s t a n t i a l p r o j e c t i o n s of N A - c o n t a i n i n g fibres a n d 5 - H T fibres from t h e locus c e r u l e u s 21 a n d r a p h e nuclei, respectively 2'2°. It has b e e n r e p o r t e d that D A d e c r e a s e d t h e s p o n t a n e o u s activity o f h i p p o c a m p a l p y r a m i d a l cells o r d e p r e s s e d g l u t a m a t e - i n d u c e d firing 3. B o t h N A a n d 5 - H T t e n d to s u p p r e s s s p o n t a n e o u s or g l u t a m a t e - i n d u c e d firing of
p y r a m i d a l cells a n d g r a n u l e s cells in t h e h i p p o c a m pus 34'19. A significant b o d y o f e v i d e n c e suggests t h a t s e r o t o n i n ( 5 - H T ) m a y play an i m p o r t a n t role in t h e m e d i a t i o n of t h e p r o c e s s e s u n d e r l y i n g l e a r n i n g a n d m e m o r y 28,29. 5 - H T a p p e a r s to inhibit t h e firing of c h o l i n e r g i c n e u r o n s within t h e h i p p o c a m p u s 23'31. A b n o r m a l f u n c t i o n i n g of t h e s e r o t o n i n e r g i c system has b e e n i m p l i c a t e d in d e m e n t i a o f t h e A l z h e i m e r type 8'9. P a t h o l o g i c a l c h a n g e s in t h e s e r o t o n i n e r g i c r a p h e syst e m m a y i m p a i r m e m o r y 41. It is well e s t a b l i s h e d t h a t v i t a m i n E scavenges t h e p e r o x y r a d i c a l in lipid p e r o x i d a t i o n 11. In r e c e n t y e a r s it has b e c o m e clear t h a t v i t a m i n E is i m p o r t a n t for norm a l n e u r o l o g i c a l f u n c t i o n 26'18. O x y g e n - d e r i v e d free r a d i c a l s a r e highly reactive c h e m i c a l species which, as well, have b e e n i m p l i c a t e d in t h e n e u r o l o g i c a l s e q u e l a e a s s o c i a t e d with v i t a m i n - E deficiency 16. F r e e radicals a l o n e m a y i n d u c e cell d e a t h by d a m a g i n g lipids or p r o t e i n s , while r e a c t i o n s b e t w e e n free radicals a n d
Correspondence: A. Machado, Departamento de BioqMmica, Bromatologla y Toxicologla, Facultad de Farmacia, Universidad de Sevilla, c/o Prof.'Garcla Gonz~lez, s/n, 41012 Sevilla, Spain. Fax: (34) (5) 423 37 65. * Recipient of a predoctoral fellowship from the Junta de Andalucla. ** Recipient of a postdoctoral fellowship from the Junta de Andalucia.
155 neurotransmitters may lead to formation of endogenous neurotoxin 4°. It has been reported that indolamines and catecholamines act as free radical scavengers 39. Interaction of neurotransmitters and free radicals may result in formation of endogenous neurotoxins. A serotoninergic neurotoxin, 5,7-dihydroxytryptamine, was reported to be formed by free radical oxidation of 5-HT in vitro 44. Moreover, it has been reported that 5-HT is oxidized by the Fenton reaction or by hypoxantine-xantine oxidase-mediated reaction in vitro, it is transformed into tryptamine-4,5-dione (4,5-DKT) 39. Little is known about the action of free radical scavengers on the neurotransmitters. The purpose of this study was to examine what effect a-tocopherol deficiency might have on the turnover of the monoamines and 5-HT 1 receptor in the rat hippocampus. We have used non-isotopic methods to estimate the turnover rate of DA, N A and 5-HT.
solvent mixtures were filtered by vacuum through a 0.45 /xm filter, and degassed by vacuum prior to ultrasound bath in order to eliminate air bubbles, which interfere with the electrochemical assay. Stock standards were prepared by accurately weighing 4 mg of each of the standard and dissolving in 4 ml 0.1 M perchloric acid and 1 m M sodium bisulfite. The stock was then stored at + 4 ° C for 2 months. The brain tissue was homogenized in 0.1 M perchloric acid containing 1 m M sodium bisulfite by ultrasonic disintegration over ice using a Labsonic 1510. Samples were centrifuged at 30,000 g for 15 min at + 4°C and the supernatant was then filtered through a 0.2 /xm filter. Material was injected by high-pressure injection valve (Rheodine), with a 20 /xl sample loop. Analyses were performed in the isocrafic mode, at a flow rate of 0.8 m l / m i n and at room temperature. Concentrations in brain samples were calculated with the aid of (linear) calibration curves obtained after the injection of pure standard. To identify small amounts in chromatograms when compounds are found near the detection limit, there are, in general, several possible ways to confirm the identity of these compounds. We carried out: Chromatograms of both a purified brain extract and a standard solution recorded at various oxidation potentials. The authentic and studied compounds should have similar electrochemical behaviour. The electrochemical behaviour of endogenous, e.g., D A and 3-MT, isolated from the brain tissue, were compared with corresponding standard solutions. The similarity of the voltagrams supports the specificity of the assay.
MATERIALS AND METHODS
NA, NMN, DA, 3-MT and 5-HT accumulation after MAO inhibition.
Animals, treatment and dissection . A group of 60 female rats of the Wistar strain weighing 200-250 g were used: 36 for m e a s u r e m e n t of turnover and 24 for binding experiments. Rats were fed semisynthetic diets containing the following vitamin E concentrations: (i) Basal diet, 170 m g / k g (Panlab S.L.); (ii) Vitamin E-deficient diet, less than 15 m g / k g (Panlab S.L.). Animals were housed two per cage, with a 12 h l i g h t / d a r k cycle, and with free access to the diets and to water. The animals were regularly weighed and their food consumption recorded. After 15 days of being fed the vitamin E-deficient diet, they were sacrificed by decapitation between 10.00 h and 11.00 h and the brains quickly removed. A cut was m a d e through the corpus callosum and the cortex was peeled back exposing the hippocampus which was carefully removed and frozen in liquid N 2 until analysis. The total time for the isolation of the tissues was less than 3 min. Pargyline HCI (75 m g / k g ) , was dissolved in 0.9% NaCI and injected intraperitoneally. Injection volume was 5 m l / k g . Products. [3H]5-hydroxytryptamine creatine sulphate (23.4 C i / m m o l ) was purchased from New England Nuclear. Pargyline HCL, 5-HT, 5HIAA, NA, NMN, D A and 3-MT were purchased from Sigma (St. Louis, MO).
Measurement of turnover Assay procedure. Analysis was performed by m e a n s of H P L C equipped with a Kontron 420 p u m p in conjunction with electrochemical detector (Bioanalytical Systems, LC-4B). Measurement of biogenic amines and their metabolites in brain. The mobile phase was prepared according to DiBussolo et al. 1°. The
NA, NMN, DA, 3-MT and 5-HT levels were determined at various times (0-20 min) after intraperitoneal injection of pargyline. NA, NMN, DA, 3-MT and 5-HT accumulation rates were obtained by calculating the slope (b) of the curve by linear regression. In this case, the standard deviation of each accumulation rate equalled standard deviation of b (SDh). Slopes of the regression lines of control and vitamin E-deficient animals were compared using the Student's t-test. Turnover of acid metabolites; DOPAC, HVA and 5-HIAA. Concentrations of D O P A C , H V A and 5 - H I A A were determined at various times (0-20 rain) after intraperitoneal injection of pargyline (75 m g / k g ) . From a semilog plot of D O P A C , H V A and 5 - H I A A concentrations versus time the slope of decline was obtained using linear regression. The slopes (b) of the regression lines were calculated by the m e t h o d of least squares. The slopes obtained were used to estimate total D O P A C , H V A and 5 - H I A A turnover rate (T) as the product of fractional rate constant (k) k = b / 0 . 4 3 4 and the estimated concentration of each metabolite at time 0 (steady state) 5. Standard deviation of k (SDk) equalled standard deviation for b (SD b) divided by 0.434. Slopes of the regression lines and the corresponding turnover rates of control and vitamin E-deficient animals were compared using the Student's t-test. 5-HT1 receptor binding assay. 5-HT 1 sites were measured by a modification of the procedure of Gozlan et al. 15. Saturation binding analyses were conducted with 9 concentrations of [3H]5-HT (0.5-15 nM) with non-specific binding estimated by competition with 10 p.M of 'cold' 5-HT. M e m b r a n e s were prepared from frozen tissue by homogenization (10 s Polytron, setting 5) in 10 vol of 50 m M Tris-HC1 buffer, pH 7.4
TABLE I
Turnover of DA, 3-MT, DOPAC, NA and NMN in hippocampus of controls and vitamin E-deficient rats after treatment with pargyline Results are given as mean_+ SD of 6 determinations. Statistical significance: * P < 0.01 low vitamin E-deficient rats vs. controls (Student's t-test).
Metabolite DA 3-MT DOPAC NA NMN
Steady state (pmol / g)
Turnover (pmol. g. h 1)
K (h 1)
Control
Low vit. E
Control
Low vit. E
Control
Low vit E
106.8_+ 31.9 62.2_+ 12.7 85.3+ 13.9 1 231.4 -+ 185.1 77.5-+ 6.8
180.4_+ 19.5 * 56.4+_ 30.4 73.1-+ 8.4 904.8 _+116.8 * 144.3_+ 35.5 *
2.7+_0.3 -
2.5_+0.3 -
469.7-+ 60.4 103.9_+ 20.7 230.3_+ 62.6 686.3 -+ 299.3 299.4_+ 29.9
1038.0_+ 166.1 * 122.9_+ 60.3 184.2_+ 17.4 641.5 _+210.6 486.5_+ 95.9 *
156 T A B L E Il
TurnoL'er of 5-HT and 5-HIAA in hippocampus of controls" and uitamin E-deficient rats after treatment with pargyline
Results are given as mean_+SD of six determinations. Statistical significance: * P < 0.05; * * P < 0.01 low vitamin E-deficient rats vs. controls (Student's t-test). Metabolite
Steady state (pmol / g)
5-HT 5-HIAA
Turnot;er (pmol. g" h - 1)
K (h - 1)
Control
Low L,it. E
Control
Low uit. E
Control
Low uit. E
1500.3 + 167.9 1319.2 _+196.5
1042.8 _+120.8 * * 843.2 + 112.2 * *
1.4 + 0.2
1.1 _+0.2 *
3 228.4 + 286.0 1846.9 +_552.2
3 428.1 _+346.9 885.4 + 269.5 * *
containing 4 nM CaC12, 1 mM EDTA and 0.1 mM PMSF. Membranes were pelleted by centrifugation (15 rain at 40,000 g). The resulting pellet was washed twice more before resuspension in 10 vols. of 50 mM Tris-HCl, pH 7.4, containing 4 mM CaCI2, 1 mM EDTA, 10 IzM pargyline and 0.1% ascorbic acid. The assay was initiated by addition of 50 /~1 of the membrane suspension (equivalent to 0.25-0.3 mg prot) to tubes containing [3H]5-HT and proceeded for 30 rain at 23°C. Following incubation, the assay mixture were rapidly filters under vacuum through glass microfiber filters (GF/B) with two 5-ml washes using 50 mM Tris-HCl buffer (pH 7.4). Dried filters were counted by liquid scintillation spectroscopy. Data analysis. The Scatchard transformation of the saturation curves were adjusted using the program LIGAND 27. The comparison between the means of different groups of data was evaluated by a Student's t-test. When the P-value was smaller than 0.05 (*) or smaller than 0.01 (**), the difference was considered significant.
inhibition by pargyline. T h e fractional rate c o n s t a n t an d the c o r r e s p o n d i n g t u r n o v e r did n o t c h a n g e bet w e e n b o t h g r o u p s of animals.
Accumulation rate o f 5-HT and turnover o f 5-HIAA. T h e 5 - H T and 5 - H I A A steady-state levels w e r e l o w e r in v i t a m i n E - d e f i c i e n t rats t h a n in controls (30.5% and 36.1%, respectively, P < 0.01; T a b l e II). A f t e r pargyline t r e a t m e n t 5 - H T s h o w e d in controls a l i n e a r accumulation
and
5-HIAA
an
exponential
declination
t h r o u g h the t i m e intervals st u d i ed (Figs. 2 A a nd 2B, respectively).
80C A
RESULTS
Body weight gain and food intake.
Body w e i g h t gain
B E Q.
400
. / ~
and food intake did not vary significantly b e t w e e n t h e control an d v i t a m i n E - d e f i c i e n t animals, suggesting that
2o0
the deficiency of v it a m i n E for 15 days was n o t detri-
o
m e n t a l to their growth. F o o d intake: controls, 16.1 _+ 2.7
3000
weight
250C
controls,
30.0_+ 4.8
g . (animal)
days)-~; v i t am i n E - d e f i c i e n t animals, ( a n i m a l s ) - 1. (15 d a y s ) - 1.
g.
Turnover o f monoamines Accumulation rate of DA, 3-MT, NA, N M N and turnover of DOPAC. A f t e r pargyline t r e a t m e n t , D A , 3-MT, N A
6
2'0
B
1 .(15
33.4_+7.8
~ rain
g d i e t . (animal) - 1 . (day)-~; v i ta m in E - d e f i c i e n t animals, 16.8 _+ 0.3 g diet • ( a n i m a l ) - 1 . (day)-~. Body gain:
~
~
200C
,< x
100C 5OO 00
' 10
2'0 rain
and N M N showed a linear a c c u m u l a t i o n t h r o u g h the 50C
t i m e intervals s t u d ie d in b o t h groups of animals (Figs.
C
1A for D A , 1B for N A and 1C for N M N ) . T h e D A
40C
steady-state c o n c e n t r a t i o n o f v i t a m i n E - d e f i c i e n t rats E
i n c r e a s e d with r e s p e c t to t h e controls (69.0%, P < 0,01)
o_
along with the D A a c c u m u l a t i o n rate (120.9%, P <
z
0.01; T a b l e I).
z
300
2OO 100 "
N o significant d i f f e r e n c e s w e r e f o u n d in the accum u l a t i o n rates of 3 - M T and N A b e t w e e n b o t h groups of animals (Table I). O n the contrary, v i t a m i n E- d ef i cient rats s h o w e d h i g h e r a c c u m u l a t i o n rate of N M N t h a n the controls (62.5%, P < 0.01; T a b l e I). DOPAC
levels d e c l i n e d e x p o n e n t i a l l y after M A O
00
i
1~3
20
_
rain
Fig. 1. Concentration of DA (A), NA (B) and NMN (C) in adult rat hippocampus of controls (open circles) and vitamin E-deficient animals (filled circles) at various times after MAO inhibition by pargyline (75 mg/kg). Each point is the mean _+S.D. of six determinations.
157 5-HT1 receptor binding. Fig. 3 shows the Scatchard analysis of [3H]5-HT binding to control and deficient vitamin E rat hippocampal membranes. The Scatchard was linear showing a single high-affinity in both groups of animals ( K a = 2.97 _+ 0.57 nM for controls and 3.41 _+ 1.17 nM for vitamin E-deficient rats). However the Bma x of vitamin E-deficient rats was higher (25.5%, P < 0.01) than in controls. Bmax w e r e : 126.7_+ 12.2 f m o l / m g prot in control rats and 159.0 _+ 11.5 f m o l / m g prot in vitamin E-deficient rats.
3000 A 250C 03 E 200C Q. I-]:
150C
t
I00C OI
o
~b
2'o rain
DISCUSSION
E .< .<
1000 800
to
d o
~b
23 rain
Fig. 2. Concentration of 5-HT (A) and semilogarithmic plot of concentration of 5-HIAA (B) in adult rat hippocampus of controls (open circles) and vitamin E-deficient animals (filled circles) at various times after MAO inhibition by pargyline (75 mg/kg). Each point is the mean _+S.D. of six determinations.
No significant changes were found in the accumulation rate of 5-HT between both groups of animals (Table II). With respect to 5-HIAA, its fractional rate constant ( K ) decreased (21.4%; P < 0.05) along with the resulting turnover (52.1%; P < 0.01) comparing deficient vitamin E with control values (Table II) 5OO 400 . ~ , , , ~ e •
LL
o controt ~
• vit, E
rn 30O
2OO
IOO
3(
50 (3H)-5HT
1OO
150
bound (fmol/mg prot)
Fig. 3. Scatchard analysis of [3H]-3HT binding activity in control (open circles) and vitamin E-deficient (filled circles) hippoeampal
membranes. Results are representative of six experiment done in duplicate. Lines were drawn by linear regression using the parametres (dissociation constant and Bmax) calculated by the program LIGAND.
Vitamin E-deficient rats showed a significant increase in hippocampus DA accumulation rate compared with control animals. On the contrary, NA and 5-HT accumulation rates were unchanged but there was a significant decrease of 5-HT and NA levels. There are several reasons to assume that all these changes were induced by the vitamin-E deficiency: (i) both diets, standard and vitamin E-deficient, were supplied by the same manufacturer, ensuring identical diet composition except in vitamin-E content; (ii) there were no differences in the food intake and corporal weight gain between groups, showing identical palatability in both diets; and (iii) it has been reported previously that similar levels of vitamin-E deficiency to that studied by us induced a significant decrease in vitamin-E content in plasma 39. Although the amount of D A in the hippocampus of the rat is only 10% of hippocampal NA, direct assessment of the DA turnover rate, by measuring DA accumulation after monoamine oxidase inhibition or the rate of D O P A C disappearance, indicated that D A is more quickly metabolized in the hippocampus than in other brain areas, including striatum 42. Therefore, due to this fast turnover rate in mesohippocampal DA, the hippocampus could be affected by free radicals in a different way to other brain structures. Some observers believe that the breakdown products of DA, which include peroxides that eventually generate cytotoxic free radicals, might in fact be the cause of tissue degeneration 3°. If this is so, then the increased DA turnover as consequence of vitamin-E deficiency would increase the risk of cell damage. In spite of an evident lack of enough data, it appears that D A can influence the excitability of hippocampal cells and thus may function as a neurotransmitter or a neuromodulator 3. After vitamin-E deficiency the NA accumulation rate was unchanged, but the NMN turnover was increased with respect to the controls. This increase in catabolism would be the cause of the decrease of NA levels after
158 vitamin-E deficiency. It has been reported that N A modulates the release of glutamate from dentate but not from C A J C A 3 of the hippocampus 22. Therefore, the above paper and another 37 have suggested that this structure may possess greater regional heterogeneity with respect to synaptic plasticity and neuromodulation than has previously been appreciated. It is interesting that vitamin-E deficiency during a short period produced a significant decrease in the 5-HT levels (30.5%). However, even though there was no significant difference in the 5-HT accumulation rate, the 5 - H I A A turnover decreased significantly. One possible way to explain these results would be that, since the 5-HT synthesis does not change and the 5-HT levels decrease, the serotoninergic fibers could have either increased its release or decreased its uptake capacity. Consequently, there was an increase of 5-HT in the synaptic cleft. As the deficit of vitamin E produces an increase of free radicals, part of this free 5-HT may be oxidized into a neurotoxic substance, such as tryptamine-4,5-dione (4,5-DKT) with a consequent decrease of catabolism. The increase of 5-HT release could be due to the action of the oxidative products. It has been reported that 4,5-DKT increases the release of 5-HT without affecting the release of D A 6. Recent experiments have provided evidence supporting the hypothesis that extracellular accumulation of synaptically released neurotransmitters, such as serotonin, have been thought to participate in the development of delayed neuronal death ~3, which could be due to the products of oxidation of 5-HT. When the 4,5-DKT, a partially oxidized form of serotonin, was injected directly into the hippocampus it produced axon terminal degeneration in sectors CA 1 and C A 3 and in the dentate gyrus 7. Moreover, it has been reported that partially oxidized serotonin (tryptamine4,5-dione) exhibits a striking propensity for medial limbic system structures especially hippocampal, entorhinal and cingulate cortices 7. To go deeper into the effect of vitamin E on 5-HT, we have studied 5-HT 1 receptors, since within the hippocampus they are the major class of serotonin receptor, and have high affinity for serotonin and its agonists 4'25. Moreover, in post-synaptic neurons, the levels of most neurotransmitter receptors are regulated in response to transynaptic activity. Receptor supersensitization (upregulation) is an adaptive response of the post-synaptic neuron to the functional loss of pre-synaptic input. Our results show that the deficit of vitamin E increased the number of 5-HT 1 binding sites in the hippocampus without affecting receptor affinity. It has been reported that serotonin receptors (5-HTa and
5-HT 2) are more resistant to modulation than their dopaminergic counterparts and that the mechanism for their modulation may be more complex for serotoninergic systems z4. Denervation lesions of serotoninergic input with 5,7-dihydroxytryptamine do not increase 5H T z binding sites 35'43. Only drugs which primarily increase serotonin concentration in the synaptic cleft are effective in down-regulation 5-HT 1 receptor binding sites 32,33A4,12. Therefore, reduced levels of serotonin could result in an up-regulation of 5-HT 1 receptors. Alternatively, there could be alterations in serotoninergic synapses which produce the increase in 5-HT1 receptors. Several aspects of the changes in 5-HT 1 receptors as a result of vitamin-E deficiency provide interesting correlations with the important modulatory role ascribed to the 5-HT in hippocampus. There is evidence to support of an important neuromodulatory role for 5-HT in the mediation of learning and memory within the hippocampus 1. This data suggests that a deficiency of vitamin E during short periods leads to significant changes of D A and 5-HT metabolism. Recently, it has been reported that vitamin E prevented CA 1 pyramidal cell death following ischemia in the gerbil 17. An understanding of the mechanism of serotoninergic neurotoxicity would allow possible high-risk brain structures to be evaluated and protective therapeutic treatments to be assessed. This work was supported by a grant from the Fundaci6n Ram6n Areces.
Acknowledgements.
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