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The effects of thiamin and its phosphate esters on dopamine release in the rat striatum
Neuroscience Letters, 158 (1993) 229-231
229
© 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00 NSL 09733
The effects of thiamin and its phosphate esters on dopamine release in the rat striatum Hiroshi Yamashita, Yu-Xiang Zhang and Shigenobu Nakamura Third Department of lnternal Medicine, Hiroshima University School of Medicine, Hiroshima (Japan)
(Received 1 April 1993; Revised version received 10 May 1993; Accepted 11 May 1993) Key words: Striatum;Dopamine; Thiamin; Thiamin triphosphate; In vivo microdialysis
The effect of thiamin and its phosphate esters on dopamine (DA) release was examined in the rat striatum using an in vivo microdialysis. Intrastriatal administration of thiamin triphosphate (TTP) or thiamin diphosphate (TDP) induced DA release, but thiamin monophosphate (TMP) or thiamin did not show any change. In the absence of Ca2+in the perfusate, TTP did not increase the DA release, to-Conotoxindid not decrease the TTP-dependent DA release. These findings suggest that, in contrast to TMP and thiamin, TTP and TDP may play a specificrole in DA release from nerve terminals.
Although thiamin deficiency has been known to cause encephalopathy, the pathogenesis involved has not been clarified. The function of this vitamin has been recognized as a coenzyme of the enzymatic system in carbohydrate metabolism [11]. C o m p a r e d with other vitamins, deficiency o f thiamin induces neurological symptoms more easily. I t o k a w a and Cooper [5, 6] have reported that thiamin has a specific role in the nervous tissue, which is independent of its coenzyme function. The role of thiamin in the brain might be related to neurotransmission besides the role as a coenzyme [2]. The locus coeruleus and dorsal m o t o r vagus, the A6 and A2 catecholamine cell groups, are frequently damaged in the brain of Wernicke-Korsakoff patients. The concentration of catecholamine metabolites in the CSF of Wernicke-Korsakoff patients is significantly reduced [9]. These reports suggest that thiamin participates in the activation of the dopaminergic nervous system. In this study, we examined the effect of thiamin and its phosphate esters, thiamin m o n o p h o s p h a t e (TMP), thiamin diphosphate (TDP) and thiamin triphosphate (TTP) on dopamine (DA) release in the striatum of rat brain using in vivo microdialysis. Anesthetized male Wistar rats (250-300 g, Hiroshima experimental animals, Japan) were placed in a stereotaxic apparatus for the implantation of a microdialysis Correspondence." H. Yamashita, Third Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minamiku, Hiroshima 734, Japan. Fax: (81) (82) 505-0490.
guide cannula. A hole (at 0 m m caudal to bregma, 3 m m lateral to bregma) was drilled through the skull to expose the dura [10]. A small slit in the dura was made so that the microdialysis probe could be lowered into the striaturn, 3.0 m m from the dural surface. A small screw was fixed to the skull to anchor the probe which was then fixed with dental cement. The animals were allowed freely access to food and water for a week after surgery to recover. The dialysis probe (BDP-1-8-03, Eicom Co., Kyoto, Japan) whose tip had a 3-mm-long semipermeable membrane was inserted into the implanted guide cannula. Each out-flow sample was collected every 20 min (perfusion rate; 2.0/.tl/min). An H P L C p u m p system (EP-10, Eicom, Co.) was connected to a M A - 5 0 D S column (4.6 m m x 15 cm, Eicom, Co.) which was kept at 25°C. The detector was a glassy-carbon electrode set at +0.70 V versus a Ag+/AgCI reference electrode. The mobile phase consisted of citric acid, sodium acetate 0.1 M each (85% of volume), methanol (15% of volume), sodium octane sulfonate (160 mg/1) and E D T A (5 mg/1), p H was adjusted to 3.9. The flow rate was 1.0 ml/min. We determined the concentration of DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) with electrochemical detection. All experiments were performed in the conscious rats 7 days after the microdialysis probe had been implanted into the striatum. Thiamin and its phosphate esters were added to the Ringer solution containing 147 m M NaC1, 4 m M KCI, 2.3 m M CaC12 (pH 6.5) and perfused for 20 min. Tetrodotoxin
230
(TTX) (2/JM), calcium free Ringer solution with EGTA (0.5 raM) and v-conotoxin (m-CgTX) (100 nM) were also infused continuously via the dialysis probe. Statistics were performed using one-way repeated measures of analysis of variance (ANOVA) and Student's t-test. The average basal level of DA, DOPAC and HVA was calculated from the mean of the 3 pooled samples for 20 min prior to the administration of drugs. After a 20 min local application with 10 mM TTP, the level of DA increased with a maximal value (1400%) after two fractions (40 min). The DA level subsequently returned to the basal level 60 min after the injection. TDP induced DA release (249% of basal) that lasted 40 min, and the DA concentration slowly returned to control values. However, thiamin and TMP did not alter the extracellular DA level (Fig. 1). TTP increased the extracellular level of DA in a dose-related manner (10 pM to 10 mM) when infused into the striatum through the dialysis probe (Fig. 2). The present findings indicate that TDP and TTP enhance the release of DA, but thiamin and TMP have no such effects, suggesting the difference between the effects of TDP and TTP and, those of thiamin and TMP. TTX has been shown to block the in vivo release of DA by inhibiting voltage-dependent Na + channels (VDNC). The control group was perfused simultaneously under the same condition except for the use of normal Ringer solution. TTX significantly reduced DA release after 20-30 min (Fig. 3). Enhancement of DA release by TTP (1 mM) was absent in rats treated with
2000 B I B
~-"-O"-,I,
t
>
_o
1500
TTP TDP TMP Thiamin
200 180" 0
< a
16o
la
140'
~
120.
100
i
=
-6
i
-5 -4 Iog[TTP] (M)
-3
Fig. 2. Dose response curve for TTP-induced DA release. All values for TTP-induced DA release were calculated as a percentage of control values which were taken as the mean of three samples prior to a TTP administration (baseline). TTP was applied for 20 min. Data are mean _+ S.E.M. (vertical bars) of 6 experiments. "P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.
TTX. The TTP-induced reduction in content of HVA was also inhibited by TTX. Calcium ions were removed from the Ringer solution and 0.5 mM EGTA added in order to determine the dependence on calcium ions. The TTP-related release of DA was suppressed by removing calcium ions from the Ringer solution. To investigate whether the DA release is mediated through a voltage-sensitive calcium channel (VSCC), m-CgTX was perfused directly into the striaturn. (o-CgTX is a peptide modulator of the N-type VSCC [3]. TTP-i'elated DA release was not significantly suppressed by the addition of (o-CgTX (Fig. 4). 200-
,,¢
150"
lOOO ¢0 .Q
_
100"
5110 W .D
N 0
0
3'0
60
Time
90
120
5o
150
Fig. 1. Effect of TTP, TDP, TMP and thiamin on DA release in the rat striatum. Thiamin and its phosphate esters ( 10 raM) were applied for 20 rain (as shown with a horizontal bar) through the inlet tube of microdialysis. All values for drug-induced DA release were calculated as a percentage of control values which are taken as the mean of the three samples prior to application. Data are mean+S.E.M. (vertical bars) of 6 experiments. *P < 0.05 ANOVA and Scheffe's tests.
2~
TTX
(min) r
-60-30
|
i
0
30
Time
i
60
9'0 1 2 0 1 5 0 1 8 0
(min)
Fig. 3. Effect o f T T X (2~tM) on TTP (l mM)-induced dopamine release from rat striatum. TTX was applied for 2 h (horizontal line). Data are mean _+ S.E.M. (vertical bars) of 5 experiments. *P < 0.05 ANOVA and Scheffe's tests.
231 T h e criterion for the n e u r o g e n i c origin 0 f t h e t r a n s m i t ter p r e s e n t in the d i a l y s a t e is d e p e n d e n c e o n the c a l c i u m ions. E l e c t r i c a l l y - e v o k e d D A release is m e d i a t e d m a i n l y by calcium influx t h r o u g h the N - t y p e V S C C [4]. T h e p r e s e n t s t u d y indicates t h a t T T P - i n d u c e d D A release is sensitive to the C a 2+ c o n c e n t r a t i o n o f the p e r f u s i o n fluid. H o w e v e r , the T T P - e v o k e d release was n o t p r e v e n t e d by the c o n v e n t i o n a l dose o f a~-CgTX. Therefore, the T T P i n d u c e d D A release w o u l d p r o b a b l y occur n o t t h r o u g h the N - t y p e V S C C . T T P m a y directly influence the N a ÷ c o n d u c t a n c e . T h e D A release b y T T P s t i m u l a t i o n a p p e a r e d sensitive to T T X . Thus, the T T P - i n d u c e d release o f D A requires the V D N C . K u n z o b s e r v e d t h a t the a n t i m e t a b o l i t e , p y r i t h i amin, i n a c t i v a t e d the N a + t r a n s p o r t system in a single n o d e o f R a n v i e r , suggesting the r e l a t i o n between N a + a n d t h i a m i n [7]. T h e p r o p a g a t i o n o f an a c t i o n p o t e n t i a l m a y involve the d e p h o s p h o r y l a t i o n o f T D P o r T T P to a l l o w the early i n w a r d flow o f N a +. T T X w o u l d p r e v e n t the flow o f N a + c u r r e n t caused b y t h i a m i n p h o s p h a t e [5, 6]. T h i a m i n derivatives are a s s o c i a t e d with the p r o t e i n which shows the highest affinity for T T X a n d is p r o b a b l y the V D N C [12]. T h e V D N C purified f r o m the b r a i n has f o u r s u b u n i t s a n d is a g o o d s u b s t r a t e for c A M P - d e p e n d ent p r o t e i n kinase [8]. A s s u m i n g t h a t T T P is the substrate for p h o s p h o r y l a t i o n o f the V D N C , it w o u l d possibly e n h a n c e the N a + p e r m e a b i l i t y o f the m e m b r a n e , dep o l a r i z e the nerve cell, a n d sequentially cause a C a 2+ influx, i n d u c i n g the D A release. T T P (10 m M ) increased the m a x i m a l D A c o n t e n t (1400%) larger t h a n T D P (249%). T h e o r d e r o f p o t e n c y
200"
• 17 []
¢D QO
4)
P
DA DOPAC HVA
150 -
a -~
lOO // // // // // // // // // la'
Iii
TTP
Ca free + TTP
1
co - C g T X + "l-rP
Fig. 4. Effect of calcium ions on DA, DOPAC and HVA release induced by 1 mM TTP in the striatum, o-Conotoxin (100 riM) was also perfused directly into the striatum through the dialysis probe. Ca2+-free Ringer solution was perfused for 4 h and TTP was given 2 h later for 20 min. Release is expressed as a percentage of basal release. Data are mean + S.E.M. (bars) of 6 experiments. *P < 0.05, **P < 0.01 Student's t-test.
i n d u c i n g D A release was T T P > T D P > T M P > thiamin. A similar effect o f a d e n o s i n e nucleotides i n d u c i n g D A release has been o b s e r v e d in PC-12 cells: A T P > A D P > A M P > adenosine, which is c h a r a c t e r i s t i c o f the Pzp u r i n o c e p t o r [13]. T h e A T P - g a t e d c u r r e n t is m e d i a t e d b y P2y-like p u r i n o c e p t o r s c o u p l e d with an ion c h a n n e l perm e a b l e to c a t i o n s [1]. We are n o w u n d e r investigations w h e t h e r T T P effect on D A release is m e d i a t e d t h r o u g h P2-purinoceptor. T h e r e d u c e d D A release in the s t r i a t u m m a y occur in t h i a m i n deficiency, leading w i d e s p r e a d d e r a n g e m e n t s in n e u r o n a l n e t w o r k s . T h e present findings m a y p r o v i d e a d d i t i o n a l evidence for the f u n c t i o n a l i m p o r t a n c e o f the d o p a m i n e r g i c i n n e r v a t i o n in the s t r i a t u m in t h i a m i n deficiency.
T h e a u t h o r s t h a n k Prof. K a w a s a k i for s u p p l y i n g the TTP. This w o r k was s u p p o r t e d by a G r a n t - I n - A i d for the R e s e a r c h C o m m i t t e e o f C N S D e g e n e r a t i v e Disease, T h e M i n i s t r y o f H e a l t h a n d Welfare o f J a p a n . 1 Burnstock, G. and Kennedy, C., Is there a basis for distinguishing two types of P2-purinocepter?, Gen. Pharmacol., 16 (1985) 433440. 2 Cooper, J.R. and Pincus, J.H., The role of thiamin in nervous tissue, Neurochem. Res., 4 (1979) 223 239. 3 Dooley, D.J., Lupp, A. and Hertting, G., Inhibition of central neurotransmitter release by o-conotoxin GVIA, a peptide modulator of the N-type voltage-sensitive calcium channel. Naunyn-Schmiedeberg's Arch. Pharmacol., 336 (1987) 467470. 4 Herdon, H. and Nahorski, S.R., Investigations of the roles of dihydropyridine and og-conotoxin-sensitive calcium channels in mediating depolarisation-evokedendogenous dopamine release from striatal slices, Naunyn-Schmiedeberg's Arch. Pharmacol., 340 (1989) 3~40. 5 Itokawa, Y. and Cooper, J.R., Ion movements and thiamine in nervous tissue-I. Intact nerve preparations, Biochem. Pharmacol., 19 (1970) 985 992. 6 Itokawa, Y. and Cooper, J.R., Ion movements and thiamine in nervous tissue-II. The release of the vitamin from membrane fragments, Biochim. Biophys. Acta, 196 (1970) 274-284. 7 Kunz, H.A., Uber die Wirkung von Antimetaboliten des Aneurin auf die einzelne markhaltige Nervenfaster, Helv. Physiol. Pharmacol. Acta, 14 (1956) 411423. 8 Levitan, I.B., Phosphorylation of ion channels, J. Membr. Biol., 87 (1985) 177-190. 9 McEntee, W.J., Mair, R.G. and Langlais, P.J.. Neurochemical pathology in KorsakolTs psychosis: implications for other cognitive disorders, Neurology, 34 (1984) 648 652. 10 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, London, 1982. 11 Peters, R.A., The biochemical lesion and its historical development, Br. Med. Bull., 25 (1969) 223 226. 12 Schoffeniels, E., Dandrifosse, G. and Bettendorff, L., Phosphate derivatives of thiamin and Na ÷ channel in conducting membranes, J. Neurochem. 43 (1984) 269 271. 13 Seta, D., Ram, E. and Atlas, D., ATP receptor: a putative receptoroperated channel in PC-12 cells, J. Biol. Chem., 266 (1991) 1799017994.
229
© 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00 NSL 09733
The effects of thiamin and its phosphate esters on dopamine release in the rat striatum Hiroshi Yamashita, Yu-Xiang Zhang and Shigenobu Nakamura Third Department of lnternal Medicine, Hiroshima University School of Medicine, Hiroshima (Japan)
(Received 1 April 1993; Revised version received 10 May 1993; Accepted 11 May 1993) Key words: Striatum;Dopamine; Thiamin; Thiamin triphosphate; In vivo microdialysis
The effect of thiamin and its phosphate esters on dopamine (DA) release was examined in the rat striatum using an in vivo microdialysis. Intrastriatal administration of thiamin triphosphate (TTP) or thiamin diphosphate (TDP) induced DA release, but thiamin monophosphate (TMP) or thiamin did not show any change. In the absence of Ca2+in the perfusate, TTP did not increase the DA release, to-Conotoxindid not decrease the TTP-dependent DA release. These findings suggest that, in contrast to TMP and thiamin, TTP and TDP may play a specificrole in DA release from nerve terminals.
Although thiamin deficiency has been known to cause encephalopathy, the pathogenesis involved has not been clarified. The function of this vitamin has been recognized as a coenzyme of the enzymatic system in carbohydrate metabolism [11]. C o m p a r e d with other vitamins, deficiency o f thiamin induces neurological symptoms more easily. I t o k a w a and Cooper [5, 6] have reported that thiamin has a specific role in the nervous tissue, which is independent of its coenzyme function. The role of thiamin in the brain might be related to neurotransmission besides the role as a coenzyme [2]. The locus coeruleus and dorsal m o t o r vagus, the A6 and A2 catecholamine cell groups, are frequently damaged in the brain of Wernicke-Korsakoff patients. The concentration of catecholamine metabolites in the CSF of Wernicke-Korsakoff patients is significantly reduced [9]. These reports suggest that thiamin participates in the activation of the dopaminergic nervous system. In this study, we examined the effect of thiamin and its phosphate esters, thiamin m o n o p h o s p h a t e (TMP), thiamin diphosphate (TDP) and thiamin triphosphate (TTP) on dopamine (DA) release in the striatum of rat brain using in vivo microdialysis. Anesthetized male Wistar rats (250-300 g, Hiroshima experimental animals, Japan) were placed in a stereotaxic apparatus for the implantation of a microdialysis Correspondence." H. Yamashita, Third Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minamiku, Hiroshima 734, Japan. Fax: (81) (82) 505-0490.
guide cannula. A hole (at 0 m m caudal to bregma, 3 m m lateral to bregma) was drilled through the skull to expose the dura [10]. A small slit in the dura was made so that the microdialysis probe could be lowered into the striaturn, 3.0 m m from the dural surface. A small screw was fixed to the skull to anchor the probe which was then fixed with dental cement. The animals were allowed freely access to food and water for a week after surgery to recover. The dialysis probe (BDP-1-8-03, Eicom Co., Kyoto, Japan) whose tip had a 3-mm-long semipermeable membrane was inserted into the implanted guide cannula. Each out-flow sample was collected every 20 min (perfusion rate; 2.0/.tl/min). An H P L C p u m p system (EP-10, Eicom, Co.) was connected to a M A - 5 0 D S column (4.6 m m x 15 cm, Eicom, Co.) which was kept at 25°C. The detector was a glassy-carbon electrode set at +0.70 V versus a Ag+/AgCI reference electrode. The mobile phase consisted of citric acid, sodium acetate 0.1 M each (85% of volume), methanol (15% of volume), sodium octane sulfonate (160 mg/1) and E D T A (5 mg/1), p H was adjusted to 3.9. The flow rate was 1.0 ml/min. We determined the concentration of DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) with electrochemical detection. All experiments were performed in the conscious rats 7 days after the microdialysis probe had been implanted into the striatum. Thiamin and its phosphate esters were added to the Ringer solution containing 147 m M NaC1, 4 m M KCI, 2.3 m M CaC12 (pH 6.5) and perfused for 20 min. Tetrodotoxin
230
(TTX) (2/JM), calcium free Ringer solution with EGTA (0.5 raM) and v-conotoxin (m-CgTX) (100 nM) were also infused continuously via the dialysis probe. Statistics were performed using one-way repeated measures of analysis of variance (ANOVA) and Student's t-test. The average basal level of DA, DOPAC and HVA was calculated from the mean of the 3 pooled samples for 20 min prior to the administration of drugs. After a 20 min local application with 10 mM TTP, the level of DA increased with a maximal value (1400%) after two fractions (40 min). The DA level subsequently returned to the basal level 60 min after the injection. TDP induced DA release (249% of basal) that lasted 40 min, and the DA concentration slowly returned to control values. However, thiamin and TMP did not alter the extracellular DA level (Fig. 1). TTP increased the extracellular level of DA in a dose-related manner (10 pM to 10 mM) when infused into the striatum through the dialysis probe (Fig. 2). The present findings indicate that TDP and TTP enhance the release of DA, but thiamin and TMP have no such effects, suggesting the difference between the effects of TDP and TTP and, those of thiamin and TMP. TTX has been shown to block the in vivo release of DA by inhibiting voltage-dependent Na + channels (VDNC). The control group was perfused simultaneously under the same condition except for the use of normal Ringer solution. TTX significantly reduced DA release after 20-30 min (Fig. 3). Enhancement of DA release by TTP (1 mM) was absent in rats treated with
2000 B I B
~-"-O"-,I,
t
>
_o
1500
TTP TDP TMP Thiamin
200 180" 0
< a
16o
la
140'
~
120.
100
i
=
-6
i
-5 -4 Iog[TTP] (M)
-3
Fig. 2. Dose response curve for TTP-induced DA release. All values for TTP-induced DA release were calculated as a percentage of control values which were taken as the mean of three samples prior to a TTP administration (baseline). TTP was applied for 20 min. Data are mean _+ S.E.M. (vertical bars) of 6 experiments. "P < 0.05, **P < 0.01, ***P < 0.001 by Student's t-test.
TTX. The TTP-induced reduction in content of HVA was also inhibited by TTX. Calcium ions were removed from the Ringer solution and 0.5 mM EGTA added in order to determine the dependence on calcium ions. The TTP-related release of DA was suppressed by removing calcium ions from the Ringer solution. To investigate whether the DA release is mediated through a voltage-sensitive calcium channel (VSCC), m-CgTX was perfused directly into the striaturn. (o-CgTX is a peptide modulator of the N-type VSCC [3]. TTP-i'elated DA release was not significantly suppressed by the addition of (o-CgTX (Fig. 4). 200-
,,¢
150"
lOOO ¢0 .Q
_
100"
5110 W .D
N 0
0
3'0
60
Time
90
120
5o
150
Fig. 1. Effect of TTP, TDP, TMP and thiamin on DA release in the rat striatum. Thiamin and its phosphate esters ( 10 raM) were applied for 20 rain (as shown with a horizontal bar) through the inlet tube of microdialysis. All values for drug-induced DA release were calculated as a percentage of control values which are taken as the mean of the three samples prior to application. Data are mean+S.E.M. (vertical bars) of 6 experiments. *P < 0.05 ANOVA and Scheffe's tests.
2~
TTX
(min) r
-60-30
|
i
0
30
Time
i
60
9'0 1 2 0 1 5 0 1 8 0
(min)
Fig. 3. Effect o f T T X (2~tM) on TTP (l mM)-induced dopamine release from rat striatum. TTX was applied for 2 h (horizontal line). Data are mean _+ S.E.M. (vertical bars) of 5 experiments. *P < 0.05 ANOVA and Scheffe's tests.
231 T h e criterion for the n e u r o g e n i c origin 0 f t h e t r a n s m i t ter p r e s e n t in the d i a l y s a t e is d e p e n d e n c e o n the c a l c i u m ions. E l e c t r i c a l l y - e v o k e d D A release is m e d i a t e d m a i n l y by calcium influx t h r o u g h the N - t y p e V S C C [4]. T h e p r e s e n t s t u d y indicates t h a t T T P - i n d u c e d D A release is sensitive to the C a 2+ c o n c e n t r a t i o n o f the p e r f u s i o n fluid. H o w e v e r , the T T P - e v o k e d release was n o t p r e v e n t e d by the c o n v e n t i o n a l dose o f a~-CgTX. Therefore, the T T P i n d u c e d D A release w o u l d p r o b a b l y occur n o t t h r o u g h the N - t y p e V S C C . T T P m a y directly influence the N a ÷ c o n d u c t a n c e . T h e D A release b y T T P s t i m u l a t i o n a p p e a r e d sensitive to T T X . Thus, the T T P - i n d u c e d release o f D A requires the V D N C . K u n z o b s e r v e d t h a t the a n t i m e t a b o l i t e , p y r i t h i amin, i n a c t i v a t e d the N a + t r a n s p o r t system in a single n o d e o f R a n v i e r , suggesting the r e l a t i o n between N a + a n d t h i a m i n [7]. T h e p r o p a g a t i o n o f an a c t i o n p o t e n t i a l m a y involve the d e p h o s p h o r y l a t i o n o f T D P o r T T P to a l l o w the early i n w a r d flow o f N a +. T T X w o u l d p r e v e n t the flow o f N a + c u r r e n t caused b y t h i a m i n p h o s p h a t e [5, 6]. T h i a m i n derivatives are a s s o c i a t e d with the p r o t e i n which shows the highest affinity for T T X a n d is p r o b a b l y the V D N C [12]. T h e V D N C purified f r o m the b r a i n has f o u r s u b u n i t s a n d is a g o o d s u b s t r a t e for c A M P - d e p e n d ent p r o t e i n kinase [8]. A s s u m i n g t h a t T T P is the substrate for p h o s p h o r y l a t i o n o f the V D N C , it w o u l d possibly e n h a n c e the N a + p e r m e a b i l i t y o f the m e m b r a n e , dep o l a r i z e the nerve cell, a n d sequentially cause a C a 2+ influx, i n d u c i n g the D A release. T T P (10 m M ) increased the m a x i m a l D A c o n t e n t (1400%) larger t h a n T D P (249%). T h e o r d e r o f p o t e n c y
200"
• 17 []
¢D QO
4)
P
DA DOPAC HVA
150 -
a -~
lOO // // // // // // // // // la'
Iii
TTP
Ca free + TTP
1
co - C g T X + "l-rP
Fig. 4. Effect of calcium ions on DA, DOPAC and HVA release induced by 1 mM TTP in the striatum, o-Conotoxin (100 riM) was also perfused directly into the striatum through the dialysis probe. Ca2+-free Ringer solution was perfused for 4 h and TTP was given 2 h later for 20 min. Release is expressed as a percentage of basal release. Data are mean + S.E.M. (bars) of 6 experiments. *P < 0.05, **P < 0.01 Student's t-test.
i n d u c i n g D A release was T T P > T D P > T M P > thiamin. A similar effect o f a d e n o s i n e nucleotides i n d u c i n g D A release has been o b s e r v e d in PC-12 cells: A T P > A D P > A M P > adenosine, which is c h a r a c t e r i s t i c o f the Pzp u r i n o c e p t o r [13]. T h e A T P - g a t e d c u r r e n t is m e d i a t e d b y P2y-like p u r i n o c e p t o r s c o u p l e d with an ion c h a n n e l perm e a b l e to c a t i o n s [1]. We are n o w u n d e r investigations w h e t h e r T T P effect on D A release is m e d i a t e d t h r o u g h P2-purinoceptor. T h e r e d u c e d D A release in the s t r i a t u m m a y occur in t h i a m i n deficiency, leading w i d e s p r e a d d e r a n g e m e n t s in n e u r o n a l n e t w o r k s . T h e present findings m a y p r o v i d e a d d i t i o n a l evidence for the f u n c t i o n a l i m p o r t a n c e o f the d o p a m i n e r g i c i n n e r v a t i o n in the s t r i a t u m in t h i a m i n deficiency.
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