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GABAB receptor activation partially inhibits N-methyl-D-aspartate-mediated tyrosine hydroxylase stimulation in rat striatal slices
European Journal of Pharmacology, 218 (1992) 335-338
335
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 21092 Short communication
GABA B receptor activation partially inhibits N-methyl-D-aspartate-mediated tyrosine hydroxylase stimulation in rat striatal slices Jos6-Antonio Arias Montafio, Daniel Martlnez-Fong and Jorge Aceves Departamento de Fisiologfa, Bioffsica y Neurocieneias, Centro de lncestigacidn y de Estudios Acanzados, Instituto Polit¢cnico Naeional de M&ico, Mexico
Received 30 March 1992,revised MS received 15 May 1992,accepted 2 June 1992
The effect of the GABA B agonist, (+)-baclofen, on the N-methyl-D-aspartate (NMDA)-mediated stimulation of tyrosine hydroxylase activity was investigated in slices of rat striatum. Tyrosine hydroxylase activity was stimulated by NMDA in a concentration-dependent manner (ECs0, 1.3 + 0.3 tzM; maximum stimulation 194 + 7% of basal activity). The action of NMDA was reversed by the NMDA antagonist, 2-amino-5-phosphonovalerate (AP-5). (+)-Baclofen (100/~M) decreased the maximum effect of NMDA by 24 + 2% without significantly modifying its ECs0. The IC50 for the inhibitory action of (+)-baclofen was 4.2 + 1.2 #M. These results show that GABA B receptor activation modulates NMDA-stimulated tyrosine hydroxylase activity, further supporting the possibility of a role of GABA in the regulation of striatal dopaminergic neurotransmission. Tyrosine hydroxylase; NMDA receptors; GABA ~ receptors; Baclofen; Dopamine synthesis; Dopaminergic transmission
I. Introduction
There is strong evidence that striatal dopaminergic function is presynaptically modulated by a number of neurotransmitters through receptors located on nigrostriatal terminals (Chesselet, 1984; Glowinski et al., 1988, and references therein). In line with this, we have recently shown that the activity of tyrosine hydroxylase (tyrosine 3-monooxygenase, E.C. 1.14.16.2), the ratelimiting step in dopamine synthesis, is modulated by glutamate and y-aminobutyric acid (GABA) by activation of presynaPtic N-methyI-D-asPartate (NMDA) and GABAB receptors respectively. Glutamate N M D A receptor activation stimulates tyrosine hydroxylase activity in a Ca2+-dependent, tetrodotoxin- and atropine-insensitive manner (Arias-Montafio et al., 1992) whereas G A B A a receptor activation inhibits the increase in ~rosine hydroxylase activity due to K+-induced depolarisation (Arias-Montafio et al., 1991). The main meg.hanism apparently underlying tyrosine hydroxylase stimulation is the entry of Ca 2+ into dopaminergic terminals through either receptor-gated
(e.g. the cationic channel present in the N M D A receptor complex) or voltage-sensitive Ca 2+ channels. On the other hand, GABA B receptors have been shown to inhibit the depolarisation-evoked Ca 2+ influx in cultured neurones and synaptosomes (Dolphin and Scott, 1986; Bowery and Williams, 1986). This mechanism is most probably involved in the GABA B receptor-mediated inhibitory effect on depolarisation-stimulated tyrosine hydroxylase activity previously reported (AriasMontafio et al., 1991). Since membrane depolarisation occurs as a result of the entry of cations through the N M D A receptor ionophore (MacDermott and Dale, 1987), we set out to study whether G A B A ~ receptor activation, by blocking Ca 2+ entry associated to depolarisation, could affect NMDA-induced stimulation of tyrosine hydroxylase activity in rat striatal slices. Enzymatic activity was assessed by measuring the accumulation of D O P A in the presence of m-hydroxybenzylamine (NSD-1015), an aromatic L-amino acid decarboxylase ( D O P A decarboxylase inhibitor).
2. Materials and methods
Correspondence to: J.A. Arias-Montafio, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK. Tel. 44.223.334045, fax 44.223.334040.
The experimental procedures have been described in detail elsewhere (Arias-Montafio et al., 1992). Briefly, male Wistar rats were killed by decapitation
336 ( + ) B a c l o f e n was p u r c h a s e d from R B I (Natick, M A , U S A ) . A l l o t h e r drugs a n d c h e m i c a l s w e r e f r o m S i g m a (St. Louis, M O , U S A ) .
200A
180" JD
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o
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3. Results 160"
140" 120'
100
-7
-6
-5
-4
Log [ N M D A ( M ) ] Fig. 1. Concentration-response curve for NMDA-mediated stimulation of tyrosine hydroxylase activity in the absence or presence of (_+)-baclofen. Striatal slices were incubated for 15 min either in the presence of NMDA (e) or, in parallel determinations, NMDA plus 100 /zM (_+)-baclofen (o), added 5 min before NMDA. The points are the combined values (means _+S.E.M.) from four experiments (triplicate determinations) and are percentages of the basal (no agonists added) enzymatic activity (2.2_+0.1 pmol DOPA min-1 mg protein-I). The line drawn is the best-fit curve obtained by fitting the data to a Hill equation. The best-fit parameters are given in the text.
a n d v i b r a t o m e - c u t striatal slices (300 p,m t h i c k n e s s a n d 3 m m d i a m e t e r discs) w e r e i n c u b a t e d at 37°C for 15 min in 1 ml K r e b s - H e n s e l e i t b u f f e r c o n t a i n i n g 5 0 0 / ~ M NSD-1015 a n d 8 /xM L-tyrosine. A f t e r t h e a d d i t i o n o f N M D A t h e i n c u b a t i o n was c o n t i n u e d for a f u r t h e r 15 min; w h e r e a p p r o p r i a t e , ( + ) - b a c l o f e n was a d d e d 5 min before NMDA. The composition of the incubation m e d i u m was (mM): NaC1 126.0; KCI 1.75; K H 2 P O 4 1.25; M g S O 4 1.0; N a H C O 3 20.2; CaC12 2.0; glucose 11.0. A t every stage of the p r o c e s s t h e m e d i u m was b u b b l e d with 5 % C O 2 in 0 2 , t h e p H b e i n g 7.4 at equilibrium. H o m o g e n a t e s o f t h e tissues w e r e c e n t r i f u g e d , a n d t h e c a t e c h o l s p r e s e n t in t h e s u p e r n a t a n t s w e r e ads o r b e d o n t o a c t i v a t e d a l u m i n a , e l u t e d with 0.4 N p e r chloric acid a n d a n a l y s e d by high p e r f o r m a n c e liquid c h r o m a t o g r a p h y ( H P L C ) c o u p l e d to e l e c t r o c h e m i c a l d e t e c t i o n . D O P A v a l u e s w e r e c o r r e c t e d a c c o r d i n g to t h e recovery of i n t e r n a l s t a n d a r d for e a c h sample. P r o t e i n c o n t e n t was d e t e r m i n e d using bovine s e r u m a l b u m i n as s t a n d a r d (Lowry et al., 1951). T h e c o n c e n t r a t i o n - r e s p o n s e d a t a w e r e f i t t e d to a Hill (logistic) e q u a t i o n using t h e H a r w e l l L i b r a r y nonl i n e a r - r e g r e s s i o n p r o g r a m m e V B O I A as i m p l e m e n t e d o n t h e C a m b r i d g e I B M 3084 ( C r a w f o r d a n d Y o u n g , 1990).
N M D A s t i m u l a t e d tyrosine hydroxylase activity in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r , r e a c h i n g a maxim u m effect o f 194 + 7 % o f b a s a l levels ( m e a n + S.E.M., four e x p e r i m e n t s ) . Best-fit values for t h e c o n c e n t r a t i o n - r e s p o n s e curves, y i e l d e d an ECs0 o f 1.3 + 0.3 p,M, a n d Hill coefficient ( n n ) , 1.1 + 0.2. T h e s t i m u l a t o r y a c t i o n of 1 0 / ~ M N M D A (191 + 5 % o f b a s a l e n z y m a t i c activity) was b l o c k e d by the s a m e c o n c e n t r a t i o n of t h e selective N M D A a n t a g o n i s t , A P - 5 (115 + 6 % o f b a s a l v a l u e s in t h e p r e s e n c e o f AP-5), giving f u r t h e r e v i d e n c e for an N M D A r e c e p t o r - m e d i a t e d effect. W h e n t h e G A B A a agonist ( + ) - b a c l o f e n (100 /zM) was a d d e d to the i n c u b a t i o n m e d i u m t h e m a x i m a l r e s p o n s e to N M D A was r e d u c e d to 165 + 6% o f b a s a l e n z y m a t i c activity b u t no significant shift in t h e ECs0, 2.0 + 0.6 p~M (fig. 1), was o b s e r v e d which was consist e n t with a n o n - c o m p e t i t i v e m o d e o f a c t i o n o f b a -
100"
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g
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80' ,< ....
Z 70
-7
-6
-5
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Log [ (+) BACLOFEN (M) ] Fig. 2. Concentration-response curve for baclofen-mediated inhibition of NMDA-stimulated tyrosine hydroxylase activity. Striatal slices were incubated with (+)-baclofen for 5 min before being exposed to 100 p.M NMDA for a further 15 min. The points are the combined values (means_+S.E.M.) from three experiments (triplicate determinations) and are percentages of the stimulation of tyrosine hydroxylase activity produced by 100/zM NMDA. Enzymatic activities were basal, 2.1_+0.1 pmol DOPA min -~ mg protein -1 and 100 p.M NMDA, 4.0+0.2 pmol DOPA min -1 mg protein I (189_+6% of basal values). The line drawn is the best curve obtained by fitting the data to a Hill equation; the best-fit values are given in the text.
337
clofen. Baclofen on its own had no effect on basal tyrosine hydroxylase activity (data not shown). The inhibition by baclofen of NMDA-mediated stimulation of tyrosine hydroxylase activity was concentration-dependent (fig. 2). The best-fit parameters for the concentration-response curve were IC50, 4.2 + 1.2 /xM; Hill coefficient (nil), 1.2 + 0.3, and 24 + 2% for the maximum attainable inhibition.
4. Discussion The results presented here show that NMDA-mediated stimulation of striatal tyrosine hydroxylase is partially inhibited by activating GABA B receptors. We have previously shown that activation of NMDA receptors increases tyrosine hydroxylase activity in a Ca 2+dependent manner (Arias-Montafio et al., 1992). This action was tetrodotoxin-insensitive, supporting a direct effect upon presynaptic NMDA receptors located on dopaminergic nerve terminals. On the other hand, tyrosine hydroxylase activity is stimulated by K+-induced depolarisation as long as Ca 2÷ is present in the bathing medium, that is, also in a Ca2÷-dependent way (El Mestikawy et al., 1985; Arias-Montafio et al., 1991). Thus the entry of the cation into dopaminergic terminals, through either voltage-activated or receptor-gated Ca 2÷ channels seems to be the main mechanism involved in the stimulation of the enzyme. The rise in intracellular Ca e÷ levels would then lead to phosphorylation and activation of tyrosine hydroxylase via Ca2+/calmodulin-dependent kinases (Yamauchi and Fujisawa, 1981). We now showed that NMDA-stimulated tyrosine hydroxylase activity was partially blocked by the GABA B agonist, (+)-baclofen, and that this inhibition appeared to be non-competitive since the effect was observed upon the maximal stimulation produced by NMDA but not on the ECs0. GABA and baclofen are able to decrease depolarisation-elicited Ca 2÷ inward currents in voltage-clamped cultured neurones (Dolphin and Scott, 1986) as well as the K+-induced rise in Ca z÷ levels in nerve terminals (Bowery and Williams, 1986), and it has been shown that GABA B receptors exert such effects by coupling to Ca z+ channels via an inhibitory G-protein (Dolphin and Scott, 1987). In line with this, we reported elsewhere that baclofen inhibited the stimulation of tyrosine hydroxylase activity caused by K+-induced depolarisation (Arias-Montafio et al., 1991), presumably by a similar mechanism, that is, by preventing the opening of voltage-sensitive Ca 2÷ channels. The entry of Ca 2+ and Na + ions through the cationic channel present in the NMDA receptor complex produces depolarisation of the membrane potential (MacDermott and Dale, 1987). This depolarisation, in turn,
might lead to he activation of voltage-sensitive Ca 2+ channels, which allows additional entry of Ca 2÷ ions, thus further increasing intracellular Ca 2÷ levels. Hence a proportion of the stimulatory effect of NMDA on striatal tyrosine hydroxylase activity could be due to the opening of voltage-activated Ca 2÷ channels. Therefore our hypothesis is that the inhibitory effect of baclofen upon NMDA-mediated tyrosine hydroxylase stimulation may be attributed to the interaction of GABA B receptors with voltage-sensitive Ca 2+ channels, blocking the opening of such channels associated with NMDA-induced depolarisation. Finally, it should be noted that the experiments described here were carried out in incubation media containing 1 mM Mg 2÷, which has been shown to block the NMDA receptor ionophore in a voltage-dependent manner. However, there is evidence that not all NMDA-mediated responses are inhibited by Mg 2÷. Thus NMDA-evoked adenosine release was observed in the presence of physiological concentrations of Mg 2+ (Hoehn et al., 1990), as was the NMDA-mediated inhibition of carbachol-induced phosphatidylinositol 4,5-biphosphate hydrolysis in slices from cortex (Gonzales, 1992) and striatum (J.A. Arias-Montafio, S. Mariscal, J. Aceves and J.M. Young, unpublished observations). Moreover, Wang (1992) reported that NMDA evoked dopamine release from striatal synaptosomes in a Mg2+-containing medium if glycine was present, a situation commonly found in brain slices (Ascher and Johnson, 1989). Whether differences in Mg 2+ sensitivity of NMDA-mediated responses reflect the existence of NMDA receptor subtypes, as has been proposed, remains to be confirmed. In summary, the results presented here show that NMDA-induced tyrosine hydroxylase activity may be modulated following agonist occupancy of GABA~ receptors, our suggestion being that this effect takes place at voltage-activated Ca 2+ channels present in dopaminergic terminals. These results give further support for a role of GABA in the control of striatal dopaminergic function.
Acknowledgements We thank Mr. A. Nuhez for excellent technical support. J.-A. Arias-Montafio was a fellow of CONACYT, M6xico.
References Arias-Montafio, J.A., D. Martlnez-Fong and J. Aceves, 1991, yAminobutyric acid (GABA B) receptor-mediated inhibition of tyrosine hydroxylase activity in the striatum of the rat, Neuropharmacology 30, 1047. Arias-Montafio, J.A., D. Martlnez-Fong and J. Aceves, 1992, Gluta-
338 mate stimulation of tyrosine hydroxylase is mediated by NMDA receptors in the rat striatum, Brain Res. 569, 317. Ascher, P. and J.W. Johnson, 1989, The NMDA receptor, its channel, and its modulation by glycine, in: The NMDA Receptor, eds. J.C. Watkins and G.L. Collingridge (IRL Press, Oxford) p. 109. Bowery, N.G. and L.C. Williams, 1986, GABA B receptor activation inhibits the increase in nerve terminals Ca 2÷ induced by depolarisation, Br. J. Pharmacol. 87, 37P. Chesselet, M.F., 1984, Presynaptic regulation of neurotransmitter release in the brain: facts and hyphotesis, Neuroscience 12, 347. Crawford, M.L.A. and J.M. Young, 1990, Potentiation by yaminobutyric acid of al-agonist-induced accumulation of inositol phosphates in slices of rat cerebral cortex, J. Neurochem. 54, 2100. Dolphin, A.C. and R.H. Scott, 1986, Inhibition of calcium currents in cultured dorsal root ganglion neurones by (-)-baclofen, Br. J. Pharmacol. 88, 213. Dolphin, A.C. and R.H. Scott, 1987, Calcium channel currents and their inhibition by (-)-baclofen in rat sensory neurones: modulation by guanine nucleotides, J. Physiol. (London) 386, 1. El Mestikawy, S., H. Gozlan, J. Glowinski and M. Hamon, 1985, Characteristics of tyrosine hydroxylase activation by K÷-induced depolarisation a n d / o r forskolin in rat striatal slices, J. Neurochem. 45, 173.
Glowinski, J., A. Ch6ramy, R. Romo and L. Barbeito, 1988, Presynaptic regulation of dopaminergic transmission in the striatum, Cell. Mol. Neurobiol. 8, 265. Gonzales, R.A., 1992, Biochemical responses mediated by N-methylD-aspartate receptors in rat cortical slices are differentially sensitive to magnesium, J. Neurochem. 58, 579. Hoehn, K., C.G. Craig and T.D. White, 1990, A comparison of N-methyl-D-aspartate-evoked release of adenosine and [3H]norepinephrine from rat cortical slices, J. Pharmacol. Exp. Ther. 255, 174. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. MacDermott, A.M. and N. Dale, 1987, Receptors, ion channels and synaptic potentials underlying the integrative function of excitatory aminoacids, Trends Neurosci. 10, 280. Wang, J.K., 1991, Presynaptic glutamate receptors modulate dopamine release from striatal synaptosomes, J. Neurochem. 57, 819. Yamauchi, T. and H. Fujisawa, 1981, Tyrosine 3 monooxygenase is phosphorylated by Ca2+-calmodulin-dependent protein kinase, followed by activation by an activator protein, Biochem. Biophys. Res. Commun. 100, 807.
335
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 21092 Short communication
GABA B receptor activation partially inhibits N-methyl-D-aspartate-mediated tyrosine hydroxylase stimulation in rat striatal slices Jos6-Antonio Arias Montafio, Daniel Martlnez-Fong and Jorge Aceves Departamento de Fisiologfa, Bioffsica y Neurocieneias, Centro de lncestigacidn y de Estudios Acanzados, Instituto Polit¢cnico Naeional de M&ico, Mexico
Received 30 March 1992,revised MS received 15 May 1992,accepted 2 June 1992
The effect of the GABA B agonist, (+)-baclofen, on the N-methyl-D-aspartate (NMDA)-mediated stimulation of tyrosine hydroxylase activity was investigated in slices of rat striatum. Tyrosine hydroxylase activity was stimulated by NMDA in a concentration-dependent manner (ECs0, 1.3 + 0.3 tzM; maximum stimulation 194 + 7% of basal activity). The action of NMDA was reversed by the NMDA antagonist, 2-amino-5-phosphonovalerate (AP-5). (+)-Baclofen (100/~M) decreased the maximum effect of NMDA by 24 + 2% without significantly modifying its ECs0. The IC50 for the inhibitory action of (+)-baclofen was 4.2 + 1.2 #M. These results show that GABA B receptor activation modulates NMDA-stimulated tyrosine hydroxylase activity, further supporting the possibility of a role of GABA in the regulation of striatal dopaminergic neurotransmission. Tyrosine hydroxylase; NMDA receptors; GABA ~ receptors; Baclofen; Dopamine synthesis; Dopaminergic transmission
I. Introduction
There is strong evidence that striatal dopaminergic function is presynaptically modulated by a number of neurotransmitters through receptors located on nigrostriatal terminals (Chesselet, 1984; Glowinski et al., 1988, and references therein). In line with this, we have recently shown that the activity of tyrosine hydroxylase (tyrosine 3-monooxygenase, E.C. 1.14.16.2), the ratelimiting step in dopamine synthesis, is modulated by glutamate and y-aminobutyric acid (GABA) by activation of presynaPtic N-methyI-D-asPartate (NMDA) and GABAB receptors respectively. Glutamate N M D A receptor activation stimulates tyrosine hydroxylase activity in a Ca2+-dependent, tetrodotoxin- and atropine-insensitive manner (Arias-Montafio et al., 1992) whereas G A B A a receptor activation inhibits the increase in ~rosine hydroxylase activity due to K+-induced depolarisation (Arias-Montafio et al., 1991). The main meg.hanism apparently underlying tyrosine hydroxylase stimulation is the entry of Ca 2+ into dopaminergic terminals through either receptor-gated
(e.g. the cationic channel present in the N M D A receptor complex) or voltage-sensitive Ca 2+ channels. On the other hand, GABA B receptors have been shown to inhibit the depolarisation-evoked Ca 2+ influx in cultured neurones and synaptosomes (Dolphin and Scott, 1986; Bowery and Williams, 1986). This mechanism is most probably involved in the GABA B receptor-mediated inhibitory effect on depolarisation-stimulated tyrosine hydroxylase activity previously reported (AriasMontafio et al., 1991). Since membrane depolarisation occurs as a result of the entry of cations through the N M D A receptor ionophore (MacDermott and Dale, 1987), we set out to study whether G A B A ~ receptor activation, by blocking Ca 2+ entry associated to depolarisation, could affect NMDA-induced stimulation of tyrosine hydroxylase activity in rat striatal slices. Enzymatic activity was assessed by measuring the accumulation of D O P A in the presence of m-hydroxybenzylamine (NSD-1015), an aromatic L-amino acid decarboxylase ( D O P A decarboxylase inhibitor).
2. Materials and methods
Correspondence to: J.A. Arias-Montafio, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, UK. Tel. 44.223.334045, fax 44.223.334040.
The experimental procedures have been described in detail elsewhere (Arias-Montafio et al., 1992). Briefly, male Wistar rats were killed by decapitation
336 ( + ) B a c l o f e n was p u r c h a s e d from R B I (Natick, M A , U S A ) . A l l o t h e r drugs a n d c h e m i c a l s w e r e f r o m S i g m a (St. Louis, M O , U S A ) .
200A
180" JD
>.I>
o
< ,.rI-
3. Results 160"
140" 120'
100
-7
-6
-5
-4
Log [ N M D A ( M ) ] Fig. 1. Concentration-response curve for NMDA-mediated stimulation of tyrosine hydroxylase activity in the absence or presence of (_+)-baclofen. Striatal slices were incubated for 15 min either in the presence of NMDA (e) or, in parallel determinations, NMDA plus 100 /zM (_+)-baclofen (o), added 5 min before NMDA. The points are the combined values (means _+S.E.M.) from four experiments (triplicate determinations) and are percentages of the basal (no agonists added) enzymatic activity (2.2_+0.1 pmol DOPA min-1 mg protein-I). The line drawn is the best-fit curve obtained by fitting the data to a Hill equation. The best-fit parameters are given in the text.
a n d v i b r a t o m e - c u t striatal slices (300 p,m t h i c k n e s s a n d 3 m m d i a m e t e r discs) w e r e i n c u b a t e d at 37°C for 15 min in 1 ml K r e b s - H e n s e l e i t b u f f e r c o n t a i n i n g 5 0 0 / ~ M NSD-1015 a n d 8 /xM L-tyrosine. A f t e r t h e a d d i t i o n o f N M D A t h e i n c u b a t i o n was c o n t i n u e d for a f u r t h e r 15 min; w h e r e a p p r o p r i a t e , ( + ) - b a c l o f e n was a d d e d 5 min before NMDA. The composition of the incubation m e d i u m was (mM): NaC1 126.0; KCI 1.75; K H 2 P O 4 1.25; M g S O 4 1.0; N a H C O 3 20.2; CaC12 2.0; glucose 11.0. A t every stage of the p r o c e s s t h e m e d i u m was b u b b l e d with 5 % C O 2 in 0 2 , t h e p H b e i n g 7.4 at equilibrium. H o m o g e n a t e s o f t h e tissues w e r e c e n t r i f u g e d , a n d t h e c a t e c h o l s p r e s e n t in t h e s u p e r n a t a n t s w e r e ads o r b e d o n t o a c t i v a t e d a l u m i n a , e l u t e d with 0.4 N p e r chloric acid a n d a n a l y s e d by high p e r f o r m a n c e liquid c h r o m a t o g r a p h y ( H P L C ) c o u p l e d to e l e c t r o c h e m i c a l d e t e c t i o n . D O P A v a l u e s w e r e c o r r e c t e d a c c o r d i n g to t h e recovery of i n t e r n a l s t a n d a r d for e a c h sample. P r o t e i n c o n t e n t was d e t e r m i n e d using bovine s e r u m a l b u m i n as s t a n d a r d (Lowry et al., 1951). T h e c o n c e n t r a t i o n - r e s p o n s e d a t a w e r e f i t t e d to a Hill (logistic) e q u a t i o n using t h e H a r w e l l L i b r a r y nonl i n e a r - r e g r e s s i o n p r o g r a m m e V B O I A as i m p l e m e n t e d o n t h e C a m b r i d g e I B M 3084 ( C r a w f o r d a n d Y o u n g , 1990).
N M D A s t i m u l a t e d tyrosine hydroxylase activity in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r , r e a c h i n g a maxim u m effect o f 194 + 7 % o f b a s a l levels ( m e a n + S.E.M., four e x p e r i m e n t s ) . Best-fit values for t h e c o n c e n t r a t i o n - r e s p o n s e curves, y i e l d e d an ECs0 o f 1.3 + 0.3 p,M, a n d Hill coefficient ( n n ) , 1.1 + 0.2. T h e s t i m u l a t o r y a c t i o n of 1 0 / ~ M N M D A (191 + 5 % o f b a s a l e n z y m a t i c activity) was b l o c k e d by the s a m e c o n c e n t r a t i o n of t h e selective N M D A a n t a g o n i s t , A P - 5 (115 + 6 % o f b a s a l v a l u e s in t h e p r e s e n c e o f AP-5), giving f u r t h e r e v i d e n c e for an N M D A r e c e p t o r - m e d i a t e d effect. W h e n t h e G A B A a agonist ( + ) - b a c l o f e n (100 /zM) was a d d e d to the i n c u b a t i o n m e d i u m t h e m a x i m a l r e s p o n s e to N M D A was r e d u c e d to 165 + 6% o f b a s a l e n z y m a t i c activity b u t no significant shift in t h e ECs0, 2.0 + 0.6 p~M (fig. 1), was o b s e r v e d which was consist e n t with a n o n - c o m p e t i t i v e m o d e o f a c t i o n o f b a -
100"
O
.,.o I- o
90
g
\÷-_÷
80' ,< ....
Z 70
-7
-6
-5
.4
Log [ (+) BACLOFEN (M) ] Fig. 2. Concentration-response curve for baclofen-mediated inhibition of NMDA-stimulated tyrosine hydroxylase activity. Striatal slices were incubated with (+)-baclofen for 5 min before being exposed to 100 p.M NMDA for a further 15 min. The points are the combined values (means_+S.E.M.) from three experiments (triplicate determinations) and are percentages of the stimulation of tyrosine hydroxylase activity produced by 100/zM NMDA. Enzymatic activities were basal, 2.1_+0.1 pmol DOPA min -~ mg protein -1 and 100 p.M NMDA, 4.0+0.2 pmol DOPA min -1 mg protein I (189_+6% of basal values). The line drawn is the best curve obtained by fitting the data to a Hill equation; the best-fit values are given in the text.
337
clofen. Baclofen on its own had no effect on basal tyrosine hydroxylase activity (data not shown). The inhibition by baclofen of NMDA-mediated stimulation of tyrosine hydroxylase activity was concentration-dependent (fig. 2). The best-fit parameters for the concentration-response curve were IC50, 4.2 + 1.2 /xM; Hill coefficient (nil), 1.2 + 0.3, and 24 + 2% for the maximum attainable inhibition.
4. Discussion The results presented here show that NMDA-mediated stimulation of striatal tyrosine hydroxylase is partially inhibited by activating GABA B receptors. We have previously shown that activation of NMDA receptors increases tyrosine hydroxylase activity in a Ca 2+dependent manner (Arias-Montafio et al., 1992). This action was tetrodotoxin-insensitive, supporting a direct effect upon presynaptic NMDA receptors located on dopaminergic nerve terminals. On the other hand, tyrosine hydroxylase activity is stimulated by K+-induced depolarisation as long as Ca 2÷ is present in the bathing medium, that is, also in a Ca2÷-dependent way (El Mestikawy et al., 1985; Arias-Montafio et al., 1991). Thus the entry of the cation into dopaminergic terminals, through either voltage-activated or receptor-gated Ca 2÷ channels seems to be the main mechanism involved in the stimulation of the enzyme. The rise in intracellular Ca e÷ levels would then lead to phosphorylation and activation of tyrosine hydroxylase via Ca2+/calmodulin-dependent kinases (Yamauchi and Fujisawa, 1981). We now showed that NMDA-stimulated tyrosine hydroxylase activity was partially blocked by the GABA B agonist, (+)-baclofen, and that this inhibition appeared to be non-competitive since the effect was observed upon the maximal stimulation produced by NMDA but not on the ECs0. GABA and baclofen are able to decrease depolarisation-elicited Ca 2÷ inward currents in voltage-clamped cultured neurones (Dolphin and Scott, 1986) as well as the K+-induced rise in Ca z÷ levels in nerve terminals (Bowery and Williams, 1986), and it has been shown that GABA B receptors exert such effects by coupling to Ca z+ channels via an inhibitory G-protein (Dolphin and Scott, 1987). In line with this, we reported elsewhere that baclofen inhibited the stimulation of tyrosine hydroxylase activity caused by K+-induced depolarisation (Arias-Montafio et al., 1991), presumably by a similar mechanism, that is, by preventing the opening of voltage-sensitive Ca 2÷ channels. The entry of Ca 2+ and Na + ions through the cationic channel present in the NMDA receptor complex produces depolarisation of the membrane potential (MacDermott and Dale, 1987). This depolarisation, in turn,
might lead to he activation of voltage-sensitive Ca 2+ channels, which allows additional entry of Ca 2÷ ions, thus further increasing intracellular Ca 2÷ levels. Hence a proportion of the stimulatory effect of NMDA on striatal tyrosine hydroxylase activity could be due to the opening of voltage-activated Ca 2÷ channels. Therefore our hypothesis is that the inhibitory effect of baclofen upon NMDA-mediated tyrosine hydroxylase stimulation may be attributed to the interaction of GABA B receptors with voltage-sensitive Ca 2+ channels, blocking the opening of such channels associated with NMDA-induced depolarisation. Finally, it should be noted that the experiments described here were carried out in incubation media containing 1 mM Mg 2÷, which has been shown to block the NMDA receptor ionophore in a voltage-dependent manner. However, there is evidence that not all NMDA-mediated responses are inhibited by Mg 2÷. Thus NMDA-evoked adenosine release was observed in the presence of physiological concentrations of Mg 2+ (Hoehn et al., 1990), as was the NMDA-mediated inhibition of carbachol-induced phosphatidylinositol 4,5-biphosphate hydrolysis in slices from cortex (Gonzales, 1992) and striatum (J.A. Arias-Montafio, S. Mariscal, J. Aceves and J.M. Young, unpublished observations). Moreover, Wang (1992) reported that NMDA evoked dopamine release from striatal synaptosomes in a Mg2+-containing medium if glycine was present, a situation commonly found in brain slices (Ascher and Johnson, 1989). Whether differences in Mg 2+ sensitivity of NMDA-mediated responses reflect the existence of NMDA receptor subtypes, as has been proposed, remains to be confirmed. In summary, the results presented here show that NMDA-induced tyrosine hydroxylase activity may be modulated following agonist occupancy of GABA~ receptors, our suggestion being that this effect takes place at voltage-activated Ca 2+ channels present in dopaminergic terminals. These results give further support for a role of GABA in the control of striatal dopaminergic function.
Acknowledgements We thank Mr. A. Nuhez for excellent technical support. J.-A. Arias-Montafio was a fellow of CONACYT, M6xico.
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