presynaptic dopamine autoreceptors control tyrosine hydroxylase activation in depolarized striatal...

Journal of Neurochemistry Raven Press, New York 0 1986 International Society for Neurochemistry

Presynaptic Dopamine Autoreceptors Control Tyrosine Hydroxylase Activation in Depolarized Striatal

Dopaminergic Terminals

S. El Mestikawy, J. Glowinski, and M. Hamon

Groupe NB, INSERM U. 114, Chaire de Neuropharmacologie, College de France, Paris, France

Abstract: The possible control of tyrosine hydroxylase (TH) activity by dopaminergic receptor-dependent mechanisms was investigated using rat striatal slices or synaptosomes incubated in the presence of various 3,4-dihydroxyphenylethylamine (dopamine or DA) agonists and antagonists. Under “normal” conditions (4.8 mM K+ in the incubating medium), the DA agonists apomorphine, 6,7-dihydroxy-N,N-dimethyl-2-aminotetralin (TL-991, 7- hydroxy-N,N-dipropyl-2-aminotetralin (7-OH-DPAT), trans- ( - ) - 4,4a,5,6,7,8,8a,9 - octahydro- 5 -propyl-2H-pyrazolo-3,4-quinoline, and 3-(3-hydroxyphenyl)-N-n-propylpiperidine decreased TH activity in soluble extracts of incubated tissues. In the case of the catechol-containing drugs apomorphine and TL-99, this effect was partly due to a direct inhibition of the enzyme, but in all other cases it appeared to depend on the stimulation of presynaptic DA autoreceptors. No effect of DA antagonists was detected on TH activity under “normal” conditions. In contrast, when tissues were incubated in a K+-enriched (60 mM) medium, (-)-sulpiride and other DA antagonists

enhanced TH activation due to depolarization whereas DA agonists were ineffective. Because ( - )-sulpiride also increased the enzyme activity in striatal slices exposed to drugs inducing release of DA, such as veratridine and d-amphetamine, it is concluded that the stimulating effect of the DA antagonist resulted in fact from the blockade of the negative control of T H normally triggered by endogenous DA acting on presynaptic autoreceptors. In contrast to TH activation due to K+-induced depolarization, the activation evoked by tissue incubation with dibutyryl cyclic AMP was unaffected by the typical agonist 7-OH-DPAT or the antagonist (-)-sulpiride. This would suggest that TH control via presynaptic DA autoreceptors normally concerns possible modulations of the cyclic AMP-dependent phosphorylation of the enzyme. Key Words: Tyrosine hydroxylase-Rat striatum-Depolar- ization-Dopamine autoreceptors. El Mestikawy S. et al. Presynaptic dopamine autoreceptors control tyrosine hydroxylase activation in depolarized striatal dopaminergic terminals. J . Neurochem. 46, 12-22 (1986).

Numerous in vivo (for review, see Roth, 1984) and in vitro (Christiansen and Squires, 1974; Iversen et al., 1976; Waggoner et al., 1980; Tissari and Gessa, 1981; Bitran and Bustos, 1982; Haubrich and Pflueger, 1982; McMillen, 1982; Saller and Salama, 1984) investigations have shown that 3,4- dihydroxyphenylethylarnine (dopamine or DA) synthesis in brain can be affected by DA agonists and antagonists, suggesting that D A receptors play some role in the control of the neurotransmitter

synthesis in dopaminergic neurons. Such control involves the first enzymatic step in the DA biosyn- thetic pathway, i.e., the formation of 3,Cdihydroxy- phenylalanine (DOPA) catalyzed by tyrosine hydroxylase (TH; L-tyrosine 3-monooxygenase; EC 1.14.16.2; Roth, 1984).

Although indirect evidence supports the proposal that some DA receptors controlling tyrosine hydroxylation in vivo are located presynaptically, on dopaminergic terminals (Roth, 1984), the most con-

~~~~~~~~ ~

Received March 5, 1985; accepted May 31, 1985. Address correspondence and reprint requests to Dr. S. El

Mestikawy at his present address: INSERM U . 288. Neurobiol- ogie Cellulaire et Fonctionnelle, Facultt de Medecine PitiC-Sal- pstritre, 91, Boulevard de I’Hbpital, F-75634 P a r i s cedex 13, France.

The present address of Dr. M. Hamon is INSERM U . 288, Neurobiologie Cellulaire et Fonctionnelle, Facultt de Mtdecine PitiC-SalpCtrikre, 91, Boulevard de I’HBpital, F-75634 Paris cedex 13, France.

Abbreviations used: DOPA, 3,4-dihydroxyphenylalanine; DA or doparnine, 3,4-dihydroxyphenylethylamine; 5-HTP, Shy- droxytryptophan; LY- I7 1555, trans-( - )-4,4a,5,6,7,8,8a, 9-octahydro-5-propyl-2H-pyrazolo-3,4-quinoline dihydrochloride; 6-MPH4, ~,~-6-rnethyl-S ,6,7,84etrahydropterin; 7-OH-DPAT, 7- hydroxy-N,N-dipropyl-2-aminotetralin; 3-PPP, 3-(3-hydroxyphenyl)-N-n-propylpiperidine; TH, tyrosine hydroxylase; TL- 99, 6,7,-dihydroxy-N,N-dimethyl-2-aminotetralin.

12

TH CONTROL BY PRESYNAPTIC DA AUTORECEPTORS 13

vincing support of this statement has been obtained in vitro, in experiments using brain slices or isolated nerve endings. For instance, Haubrich and Ptlueger (1982) reported that exposure of striatal synaptosomes to some DA agonists reduces their capacity to synthetize [3H]DOPA from [3H]tyrosine and that this effect can be reversed by DA antagonists.

To our knowledge, however, such studies con- cerned the modulation of tyrosine hydroxylation by DA agonists and antagonists, and no report has yet been published about the possible changes in the intrinsic activity of TH extracted from dopaminergic terminals incubated with these drugs. This is an important question, because previous studies have shown that elucidation of the mechanism re- sponsible for the acceleration of DA synthesis in depolarized dopaminergic terminals could be achieved mainly because depolarization is associated with alterations in the intrinsic properties of TH (Simon and Roth, 1979; El Mestikawy et al., 1983, 1985). In an attempt to explore the molecular mecha-

nisms involved in the modulation of DA synthesis via the stimulation or blockade of presynaptic DA autoreceptors, we looked therefore at the possible changes in the activity of TH extracted from striatal slices or synaptosomes exposed to various agonists and antagonists. These studies were performed not only with tissues incubated in normal Krebs-Hen- seleit medium, but also with those incubated in K+- enriched medium or in the presence of dibutyryl cyclic AMP, two conditions associated with a marked activation of TH (Simon and Roth, 1979; El Mestikawy et al., 1983). Such experiments indicated that the incubation of dopaminergic terminals with DA agonists or antagonists did induce signif- cant alterations in the intrinsic activity of TH. Fur- thermore, evidence is presented that endogenous DA itself could control TH activity via the stimulation of presynaptic DA autoreceptors.

MATERIALS AND METHODS ~[3,5-~H]Tyrosine (50 Ci/mmol; New England Nuclear)

was purified according to the method of Giorguieff et al. (1977) just before being used for TH assays.

Other compounds used were t-tyrosine (Merck), L- tryptophan (Merck), ~,~-6-methyl-5,6,7,8-tetrahydro- pterin (6-MPH4; Sigma), catalase (12,500 unitslmg; Sigma), reserpine (Ciba-Geigy), veratridine (Dr. E. Jover, INSERM U. 172, Marseille, France), dibutyryl cyclic AMP (Sigma), EGTA (Sigma), carbidopa (Ciba-Geigy), 5- hydroxytryptophan (5-HTP; Calbiochem), and d-amphetamine (Delagrange).

The DA agonists and antagonists used in the present study were apomorphine (Merck); TL-99 (6,7-dihydroxy- N,N-dimethyl-2-aminotetralin; Dr. S. Z. Langer, Syn- thelabo LERS, Paris, France); 7-OH-DPAT (7-hydroxy- N,N-dipropyl-2-aminotetralin; Dr. A. S. Horn, Gro- ningen, The Netherlands); LY-171555 [trans-( -)-

4,4a ,5,6,7,8 $a, 9 - octahydro - 5 - propyl - 2H -pyrazolo - 3,4- quinoline dihydrochloride] and lergotrile (Eli Lilly & Co.); ( + )- and ( - )-3-PPP [3-(3-hydroxyphenyl)-N-n-propylpiperidine], Ro 17-48891001, Ro 17-48901001, and methiothepin (Hoffmann-La Roche); lisuride (Schering AG); bromocriptine (Sandoz); ( +)- and ( - )-sulpiride (Delagrange); clozapine (Sandoz); haloperiodol and pimozide (Janssen); and chlorpromazine and chlorpromazine sulfoxide (RhBne-Poulenc).

In vitro incubation of brain slices Adult male Sprague-Dawley rats (Charles River strain),

weighing -250 g, were killed by decapitation, and their brains were rapidly removed in the cold (4°C). Striata, hippocampi, and cerebral cortex were dissected as described by Glowinski and Iversen (1966) and sliced (0.3 mm thickness) using a McIlwain tissue chopper. Tissues (-85 mg of striaturn, -1 10 mg of hippocampus, or -320 mg of cerebral cortex) were suspended in 5 ml of Krebs- Henseleit medium (in mM, NaCl 128, KCI 4.8, CaCI, 1.3, MgSO, 1.2, Na,HPO, 15.8, and glucose 10) continuously saturated by an atmosphere of 95% 0,/5% CO, (final pH 7.3). Samples were incubated for I5 min at 37°C in the presence of various drugs (see Results), and tissues were collected by rapid filtration through a nylon mesh (pore size 50 pn) placed on Whatman no. 3 filter paper. Within 5 s of stopping the incubation, the nylon mesh with tissues was put on dry ice, and tissues were kept frozen for no more than 1 h before homogenization. Each frozen sample was homogenized at 0°C in 1.5 ml of 20 mM Tris- HCI, pH 7.0 (striatum and hippocampus), or 50 mM Tris- acetate, pH 7.6 (cerebral cortex), using a Polytron F T 10 OD apparatus. In both cases, the homogenizing buffer was supplemented with 2 mM P-mercaptoethanol. After centrifugation (40,000 g, 20 min, 4 T ) , TH (striatum and hippocampus) or tryptophan hydroxylase (cerebral cortex) was assayed in each supernatant. In some cases, supernatants were filtered through Sephadex G-25 columns (PD 10; Pharmacia) to remove small molecules pos- sibly interfering in subsequent enzyme assays.

When incubations were performed in K+ -enriched medium (60 mM K+), the NaCl concentration was reduced to 72.8 mM to maintain constant the osrnolarity of the incubating medium. In contrast, no compensatory change was made when Ca2+ was omitted; in that case, the incubating medium was supplemented with 0.1 mM EGTA.

Experiments with crude synaptosomal fractions Pooled striata from 20 adult male rats were homoge-

nized at 4°C in 10 volumes (voVwt) of 0.32 M sucrose using a Potter-Elvehjem apparatus fitted with a Teflon pestle. The homogenate was centrifuged at 800 g for 10 min, and the supernatant was recentrifuged at 10,OOO g for 30 min. The pellet (PJ was finally suspended in 10 volumes of the same ice-cold Krebs-Henseleit medium as that used for slices.

Aliquots (0.1 ml) of this suspension were mixed with the same medium supplemented with various drugs (see Results) up to a final volume of 0.5 ml. Samples were incubated for 15 rnin at 37°C under a constant atmosphere of 95% O#% CO, and then centrifuged (8,OOO g, 10 min, 4°C). The supernatant was discarded, and the pellet was rapidly frozen on dry ice. Within < I h, each pellet was then sonicated in 0.3 ml of 20 mM Tris-HCI, pH 7.0, containing 2 mM P-mercaptoethanol. TH was assayed in du-

J . Neurochrm.. Vol. 46. No. 1 . 1986

14 S. EL MESTIKA WY E T AL.

plicate in supernatants obtained by centrifuging samples at 40,000 g for 20 min at 4°C.

TH assay The procedure originally described by Nagatsu et al.

(1961) was used with the following modifications: An aliquot (0.1-0.2 ml) of each supernatant or Sephadex G-25 eluate was mixed with 10 pM[?H]tyrosine. 2,000 units of catalase. 20 mM Tris-HCI. 2 mM P-mercaptoethanol, and 130 mM NaCH,COO, pH 7.0 (final volume 0.24 ml). The reaction was started at 37°C with the addition of 10 pl of a 6.25 mM solution of 6-MPH4 (final concentration 0.25 mM) and stopped IS min later by addition of 1 ml of 10% trichloroacetic acid. Samples were centrifuged (8,000 g, 10 min. PC), and the supernatants were passed through Dowex AG 50 WX4 (H’ form) columns (0.8 cm in di- ameter, I cm high). The column effluent and wash (1 ml of H,O) were collected in scintillation vials and mixed with 10 ml of Aquasol (New England Nuclear) for radio- activity counting of [’H]H20 formed by [3H]tyrosine hydroxylation. TH activity was expressed in nmol of [’HIDOPA formedhg of proteidl5 min.

Tryptophan hydroxylase assay The protocol for tryptophan hydroxylase assay has

been described in detail elsewhere (Hamon et al., 1979). In brief, an aliquot (0.3 ml) of the 40.000-g supernatant of each tissue homogenate was mixed with 0.15 mM L- tryptophan, 20 pM carbidopa, 2 mM P-mercaptoethanol, 2,000 units of catalase, and 0.16 mM 6-MPH4, and the reaction was allowed to proceed at 37°C for I5 min. The reaction product 5-HTP was then measured spectrofluo- rometrically, and the enzyme activity expressed as nmol of 5-HTP forrnedmg of proteidl5 min.

Protein content was determined by the Folin phenol procedure (Lowry et al., 1951) using bovine serum al- bumin (Sigma) as the standard.

Statistical calculations were performed according to the procedures of Snedecor and Cochran (1967). When the p value was >0.05 (by unpaired Student’s f test), a difference was considered to be nonsignificant.

RESULTS

Effects of tissue exposure to 7-OH-DPAT and other DA agonists on TH activity in striatal dopaminergic terminals incubated under “normal” or depolarizing conditions

Although 7-OH-DPAT did not affect T H activity when added to the 40,000-g supernatant of striatal homogenate (Fig. l) , incubation of striatal synaptosomes or slices with this drug resulted in a significant reduction of the enzyme activity extracted from the incubated tissues (Fig. 1). Maximal reduction reached 30-35% compared with T H activity from control tissues, and half-maximal effect was obtained by exposing striatal slices o r synaptosomes to -30 nM 7-OH-DPAT (Fig. 1).

As shown in Table 1, other DA agonists such as apomorphine, TL-99, LY 171555, and (+)-3-PPP also reduced T H activity when added to the “normal” (4.8 mM K + ) incubating medium of

stnatal slices. In contrast, (-)-3-PPP, lisuride, bromocriptine, and lergotrile were inactive even at a high concentration (1-10 pM) (Table 1). Because apomorphine exerted a direct inhibitory effect on TH activity in the supernatant of striatal tissues (ICso 3 1.30 p M ; see also Waggoner et al., 1980; Laschinski et al., 1984), we investigated whether the inhibition observed following incubation of slices with this drug might have resulted from its presence in the 40.000-g supernatant of tissue homogenate. Indeed, filtration of the tissue extract through a Sephadex G-25 column allowed the complete removal of apomorphine [as checked with [3H]apomorphine (data not shown)], but did not completely suppress the enzyme inhibition: A significant 25-30% reduction of T H activity still persisted in the Sephadex filtrates from tissues incubated in the presence of 1 o r 10 FM apomorphine. However, this reduction was less pronounced than that observed when TH was measured in crude supernatants: -48 and -65% with 1 and 10 FM apomorphine, respectively. The filtration of tissue extracts through Sephadex G-25 columns also resulted in some decrease in T H inhibition due to the incubation of striatal slices with 10 p M TL-99 (-24% instead of -36%). In contrast, the effect of tissue incubation with 7-OH-DPAT, LY171555, or (+)-3- PPP on T H activity remained unchanged by filtering the tissue extracts through Sephadex G-25 columns (data not shown).

As previously noted (Simon and Roth, 1979; El Mestikawy et al., 1983), the incubation of striatal slices in K+-enriched (60 mM) medium resulted in a marked activation of TH, as pronounced in the 40,000-g supernatant (Table 1) a s in the corresponding Sephadex filtrate of incubated tissues (data not shown). This activation was generally associated with a loss of sensitivity to DA agonists, because among the whole series examined, only apomorphine was still able to inhibit partially T H activity when added to the K+-enriched incubating medium of striatal slices (Table 1). However, the drug effect ( - 55%) was less pronounced than that observed under “normal” (4.8 mM K + ) conditions (-65%; Table 1). Furthermore, the effect was no longer significant when tissue extracts were filtered through Sephadex G-25 columns to remove the drug (data not shown). Data in Table 1 also indicate that ( -)-3-PPP altered TH activity in depolarized tissues, but in this case a stimulatory effect was noted.

Effects of tissue exposure to ( - )-sulpiride and other DA antagonists on TH activity in striatal dopaminergic terminals incubated under “normal” or depolarizing conditions

The incubation of striatal slices in “normal” (4.8 mM K + ) medium supplemented with 10 nM-10 p M (-)-sulpiride resulted in no change in the enzyme activity compared with that found in the 40,000-g

J . Neurochem.. Vol. 46, No. I , 1986

TH CONTROL BY PRESYNAPTIC DA AUTORECEPTORS 15

-I

\ * \\ Slices

-1 ,,

C -I - 1 - 6 - 5 -4

log [7-OH-DPAT]M

FIG. 1. Effects of 7-OH-DPAT on TH activity from rat striatum. Striatal slices (0) or crude synaptosomal fractions (P2; 0) were incubated for 15 min at 37°C in normal (4.8 mM K') medium containing various concentrations of 7-OH-DPAT. Incubated tissues were then homogenized in 20 mM Tris-HCI, pH 7.0, containing 2 mM p-mercaptoethanol and centrifuged. TH activity was measured in each supernatant and expressed as a percentage of that found in the absence (C) of 7-OH-DPAT. For comparison, TH activity in the soluble extract (&; 40,000-g supernatant of the homogenate) of control striatal slices was also measured after addition of 7-OH-DPAT (10 nM-0.1 mM) directly to the enzyme assay mixture (0). Data are mean f SEM values of four to eight independent determinations. In the absence of 7-OH-DPAT. absolute TH activities (100%) were 0.348 2 0.017 nmol of [3H]DOPNmg of protein/l5 min in experiments with striatal slices and 0.926 f 0.041 nmol of [3H]DOPN mg of protein/l5 min with crude synaptosomal P, fractions. 'P < 0.05 compared with respective control values.

supernatant of tissues incubated without the drug. In contrast, under depolarizing conditions (60 mM K+) , ( - )-sulpiride induced a concentration-dependent increase in TH activity (Fig. 2), with the maximal effect (40%) being observed with 0.1 - 10 p M drug. The (+)-enantiomer of sulpiride was also able to enhance TH activation due to K+-induced depolarization, but it was markedly less effective than (-)-sulpiride: Even at 10 p M , (+)-sulpiride produced an increase in TH activity only half that due to 0.1 p M (-)-sulpiride (Fig. 2). As previously

noted for TH activation resulting from K+-induced depolarization (El Mestikawy et al., 1989, the activation due to exposure of depolarized tissues to ( - )-sulpiride persisted entirely following filtration of tissue extracts through Sephadex G-25 columns (data not shown).

In addition to ( -)-sulpiride, several other DA antagonists had no effect on TH activity in nondepo- larized tissues, but increased the enzyme activation due to K+-induced depolarization (Table 2). This was notably the case with clozapine, haloperidol,

TABLE 1. Effects of various DA agonists on activity of TH from striatal slices incubated under control ( K + = 4.8 mM) or

depolarizing ( K + = 60 mM) conditions

TH activity (%)

Agonist [ K + ] 4.8 mM [K'] 60 mM

None

Apomorphine (10 p M ) TL-99 (10 p M )

LY 171555 (I0 p M ) 7-OH-DPAT (1 p M )

( + )-3-PPP ( 1 p M ) (-)-3-PPP ( I p M ) Lisuride (10 pM) Bromocriptine (10 p M ) Lergotrile (10 p M )

100 (0.354 t 0.024)

35.3 t 3.Y 64.2 r 6.5"

70.8 f 5.9" 73.1 f 5.3" 76.5 t 2.5' 94.6 r 3.9 88.2 f 9.4 94.1 f 4.3 91.4 t 8.3

100(0.609 t 0.041)

45.3 t 5.3" 81.7 t 9.1

92.3 t 4.1 94.2 t 5.6

101.4 f 4.2 116.8 t 4.8"

108.2 f 4.5 113.7 f 9.8

%.8 t 8.9

~~ ~~ ~

Striatal slices were incubated for I 5 min at 37°C in normal (4.8 mM K + ) or K+-enriched (60 mM) medium supplemented with various DA agonists. Tissues were homogenized in 20 mM Tris-HCI, pH 7.0, containing 2 mM p-mercaptoethanol and centrifuged. TH activity was measured in each supernatant and expressed as a percentage of that found in the absence of DA agonists (absolute control values in nmol of [3H]DOPN mg of protein/l5 min are given in parentheses). Data are mean r SEM values of at least six independent determinations.

p < 0.05 compared with respective control values (no agonist).

J . Neurochem., Vul. 46. No. I . 1986

16 S . E L MESTIKAWY ET AL.

Y I I

C - 8 -7 - 6 - 5

log [ ~ u ~ p i r i a e ] ~

FIG. 2. Effects of (+)- and (-)-sulpiride on TH activity from striatal slices incubated under depolarizing conditions. Striatal slices were incubated for 15 min at 37°C in K+-enriched (60 rnM) medium in the absence (C) or presence of various concentrations of (+)-sulpiride (0) or (-)-sulpiride (0). Tissues were homogenized in 20 mM Tris-HCI, pH 7.0, containing 2 rnM p-mercaptoethanol, and centrifuged. TH activity was assayed in each supernatant and expressed as nmol of [3H]DOPNrng of protein/l5 min. Data are mean 2 SEM values of seven indeDendent determinations. *D < 0.05

>I0 p M (data not shown). When added at 10 pM to the incubating medium, methiothepin and chlorpromazine sulfoxide did not significantly change TH activity (Table 2).

Effects of DA antagonists on reduction of TH activity in dopaminergic terminals exposed to 7-OH-DPAT

As expected from an effect of 7-OH-DPAT via the stimulation of DA receptors (Seiler and Markstein, 1984), the DA antagonist ( - )-sulpiride ( I p M ) completely prevented the reduction in TH activity from slices or synaptosomes incubated with 1 p M 7-OH- DPAT (Fig. 3).

Experiments with synaptosomes were more ap- propriate for investigating the possible concentration dependency of the reversal by antagonists of 7-OH-DPAT-induced inhibition of TH. As illus- trated in Fig. 4A, (-)-sulpiride (1 pM) produced a shift to the right of the inhibition curve with in- creasing concentrations of 7-OH-DPAT. Compar- ison of the relative activities of a series of antagonists indicated that haloperidol was more potent than ( - )-sulpiride in protecting synaptosomal TH from 7-OH-DPAT-induced inhibition. In contrast, clozapine and ( + )-sulpiride were markedly less potent in this respect (Fig. 4B).

Effects of (-)-sulpiride and other DA antagonists compared with TH activiiy in the absence of (+)-' or (-)- sulpiride.

chlorpromazine, and pimozide. In most cases, maximal activation (30-40%) was observed with 10 p.M drug, except of haloperidol, which produced a bi- phasic change in TH activity: an activation at 0.1- 1 .O p M (Table 2) and an inhibition at concentrations

on TH activity instfiatal slices incubated under various conditions stimulating or inhibiting DA release

Because (-)-sulpiride was found to increase TH activity only in striatal slices depolarized by K', from which DA release was markedly enhanced (data not shown; see also Arbilla and Langer, 1980), additional experiments were carried out to assess

TABLE 2. Effects of various DA antagonists on activity of TH from striatal slices incubated under control ( K + = 4.8 mM) or

depolarizing ( K + = 60 m M ) conditions

TH activity (5%)

Antagonist (K'] 4.8 mM (K'160 mM

None 100 (0.335 5 0.021) 100 (0.566 2 0.041) (-)-Sulpiride (10 p M ) 91.6 f 3.9 142.5 2 6.5"

Haloperidol (1 +M) 94.0 -+ 7.9 138.2 2 7.20 Chlorpromazine (10 p M ) 100.2 2 5.7 131.9 2 4.1" Chlorpromazine-SO (10 p M ) 98.7 2 4.8 110.3 f 5.1 Pimozide (10 F L M ) 108.0 5 6.9 129.3 f 6.4" Methiothepin (10 pM) 102.3 ? 10.3 119.0 f 7.4

Clozapine (I0 p M ) 91.9 f 4.8 140.9 2 1O.Y

Striatal slices were incubated for 15 min at 37°C in normal (4.8 mM K') or K+-enriched (60 mM) medium supplemented with various DA antagonists. Tissues were homogenized in 20 mM Tris-HCI, pH 7.0, containing 2 mM p- mercaptoethanol and centrifuged. TH activity was measured in each supernatant and expressed as a percentage of that found in the absence of DA antagonists (absolute control values in nmol of [3H]DOPA/mg of protein/l5 min are given in parentheses). Data are mean f SEM values of at least six independent determinations.

a p < 0.05 compared with respective control values (no antagonist).

J . Neurochem., Vol. 46, No. I , 1986

TH CONTROL BY PRESYNAPTIC D A AUTORECEPTORS 17

SLICES F--

- + - + + + - -

FIG. 3. Reversal by (-)-sulpiride of the inhibitory effect of 7-OH-DPAT on TH activity in striatal slices or synaptosornes. Striatal slices or crude synaptosomal fractions (P,) were incubated in normal medium (4.8 rnM K') containing 1 yM 7- OH-DPAT and/or 1 pM (-)-sulpiride as indicated. Tissues were then homogenized in 20 rnM Tris-HCI. pH 7.0, containing 2 mM p-mercaptoethanol and centrifuged. TH activity was measured in each supernatant and expressed as a percentage of that found in the absence of drugs. For slices, absolute TH activity was equal to 0.338 T 0.017 nrnol of [3H]DOPNmg of protein/l5 min; for P, fractions, TH activity was equal to 0.917 rt 0.046 nmol of [3H]DOPNmg of protein/ 15 min. Data are mean rt SEM values of six separate determinations. 'p < 0.05 compared with respective control ("no drug") values.

the possible influence of DA release on the acti- vating effect of the DA antagonist on this enzyme. Striatal slices were first incubated under two conditions known to induce DA release, i.e., in the presence of d-amphetamine (Arbilla and Langer, 1980) or of veratridine (Levi et al., 1980). As illus- trated in Fig. 5, tissue exposure to the depolarizing agent veratridine (10 pM) but not to d-amphetamine (5 p M ) resulted in a significant increase in TH activity. However, when (-)-sulpiride (10 p M ) was added to the incubating medium, an activation of TH was noted with the two DA release-inducing drugs (Fig. 5). Thus, TH activity increased by 30% (p < 0.05) following tissue incubation with d-amphetamine plus (-)-sulpiride (Fig. 5) and by 95% (p < 0.05) in slices exposed to veratridine plus (-)- sulpiride (instead of 41% with veratridine alone; Fig. 5).

In a second set of experiments, possible changes in TH activity due to (-)-sulpiride and other DA antagonists were explored using depolarized tissues from which DA release was, in contrast, prevented or markedly reduced. For this purpose, K+-induced deplarization was achieved either with striatal slices incubated with EGTA (0.1 mM) or with the same tissue from reserpine-treated rats. Measurement by HPLC coupled to electrochemical detection (El Mestikawy et al., 1985) of endogenous DA recov- ered in the medium at the end of the 15-min incu-

bation confirmed that in vitro addition of EGTA and in vivo reserpine treatment reduced markedly ( - 90 and - 75%, respectively) the K+-induced overflow of DA from striatal slices (data not shown).

In agreement with previous data (Simon and Roth, 1979; El Mestikawy et al., 1983), we observed that TH activation normally provoked by K+-induced depolarization disappeared completely when tissues were incubated in the absence of extracel- lular Ca2+ (Fig. 6). Similarly, the stimulatory effect of (-)-sulpiride on the enzyme activity was lost when striatal slices were depolarized in Caz+-free medium (Fig. 6).

Following reserpine treatment (10 mg/kg intraperitoneally 24 h before death), basal TH activity was already increased (60-70%), but K+-induced depolarization produced a further enhancement of the enzyme activity (Table 3; see also El Mestikawy et al., 1985). However, (-)-sulpiride, haloperidol, or methiothepin did not augment TH activation due to depolarization of striatal slices from reserpine- treated rats (Table 3), in contrast to that observed using tissues from control animals (Table 2).

Effects of 7-OH-DPAT and (-)-sulpiride on TH activity in striatal slices incubated with dibutyryl cyclic AMP

In addition to inducing DA release from striatal slices, K+-induced depolarization also enhanced DA synthesis and TH activity (Tables 1 and 2), and therefore we investigated whether the depolarization-induced changes in the effects of 7-OH-DPAT and (-)-sulpiride on the striatal enzyme (Tables 1 and 2) could be reproduced by another treatment producing TH activation, e.g., the incubation of slices with dibutyryl cyclic AMP (Simon and Roth, 1979). Previous investigations (El Mestikawy et al., 1985) have shown that maximal TH activation could be obtained by addition of 2.5 mM dibutyryl cyclic AMP to the incubating medium; in the present case, this treatment resulted in a 133% increase in the enzyme activity extracted from the incubated tissues (Table 4). As previously noted for TH activation due to K+-induced depolarization (Table I ) , the activity increase produced by dibutyryl cyclic AMP was associated with a loss of the enzyme sensitivity to the inhibitory effect of 7-OH-DPAT. However, in contrast to the activation observed with depolarized tissues, TH activation in striatal slices incubated in the presence of dibutyryl cyclic AMP was not enhanced when (-)-sulpiride was added to the incubating medium (Table 4).

Effects of tissue incubation with 7-OH-DPAT or (-)-sulpiride on TH activity in hippocampal slices

In contrast to that observed with striatal slices, neither 10 p M 7-OH-DPAT nor 10 p M (-)-sulpiride altered TH activity when added to the incubating medium of hippocampal slices. This applied to

1. Neurorhem.. Vol. 46. No. 1 . 1986

18 S. EL MESTIKAWY ET AL.

1.0 I/ I I 1.0

C - E

e ul ’5- 0.9

P

0.0 5 - 0 : 0.1 - z ? 0.6 I n

0.5 C -9 -8 -7 -6 - 5 -0

lop [7-OKDPAT] log b n t a g ~ n i s t ] ~

FIG. 4. Effects of DA antagonists on 7-OH-DPAT-induced inhibition of TH in crude synaptosomal fractions from rat striata. A: P, fractions were incubated for 15 min at 37°C in normal medium (4.8 mM K’) in the absence (0) or presence (0) of 1 pM ( - ) - sulpiride and various concentrations of 7-OH-DPAT. B: P, fractions were incubated for 15 rnin at 37°C in normal medium (4.8 mM K’) in the presence of 1 pM 7-OH-DPAT and various concentrations of DA antagonists: haloperidol (m), (-)-sulpiride (O), (+)-sulpiride (+), and clozapine (X). After incubation, synaptosomes were collected by centrifugation and sonicated in 20 mM Tris-HCI, pH 7.0, containing 2 mM p-mercaptoethanol. TH activity was measured in the 40,000-g supernatant of each homogenate and expressed in nmol of [3H]DOPNmg of proteinll5 min. Data are mean ? SEM values of triplicate determinations in three independent experiments. The ranges of TH activity values in the absence of antagonists (C) but without (A) or with (A) 1 pM 7-OH-DPAT are indicated by the stippled areas. In the absence of 7-OH-DPAT. none of the antagonists (at concentrations up to 10 FM) induced any significant change in TH activity.

“normal” (4.8 mM K+) as well as to depolarizing (60 mM K + ) conditions, although a significant activation of TH occurred in tissues incubated in K+- enriched medium (normal 22.8 2 2.0 pmol of [3H]DOPA/mg of proteidl5 min; K+-enriched 33.3 2 3.1 pmol of [3H]DOPA/mg of proteinll5 min; n = 7 in each group; p < 0.05).

Effects of tissue incubation with 7-OH-DPAT, (-)-sulpiride, or other DA antagonists on activity of tryptophan hydroxylase in cerebral cortex slices

Under normal (4.8 mM K + ) conditions, neither the addition of 10 FM 7-OH-DPAT nor that of any DA antagonist [ 10 F M ( -)-sulpiride, clozapine, chlorpromazine, or methiothepin] to the incubating medium changed tryptophan hydroxylase activity extracted from incubated cortical slices. K+-induced depolarization was associated with a marked increase in the enzyme activity (normal 0.152 c 0.005 nmol of 5-HTP/mg of proteinl15 min; K+-enriched 0.245 c 0.009 nmol of 5-HTP/mg of protein/ 15 min; n = 8 in each group; p < 0.05), which was not significantly altered when 7-OH-DPAT or a DA antagonist was added to the incubating medium (data not shown).

DISCUSSION

Although several authors (see references in the introductory section) already showed that DA agonists and antagonists can affect the rate of DA synthesis in striatal slices or synaptosomes, the present study is the first to demonstrate that pre-

synaptic DA autoreceptors control DA synthesis by regulating the intrinsic activity of the rate-limiting enzyme, TH. Indeed, DA agonist-induced reduction of DA synthesis (Christiansen and Squires, 1974; Waggoner et al., 1980; Bitran and Bustos, 1982; Haubrich and Pflueger, 1982), was associated with a significant decrease in the activity of soluble TH extracted from slices or synaptosomes exposed to these drugs.

In the case of catechol-containing agonists (apomorphine and TL-99), the reduction in TH acctivity persisted only partially after the soluble extract of incubated tissues was filtered through Sephadex G- 25 columns. Because this procedure allowed the elimination of small molecules, including the agonists added to the incubating medium (and contam- inating the tissue extract), it could be proposed that part of the reduction in TH activity resulted in fact from the direct competitive interaction between the catechol-containing drug and the pteridin cofactor (6-MPH4) of the enzyme (Laschinski et al., 1984). However, this did not occur with the three other agonists that also induced a reduction of TH activity [7-OH-DPAT, LY 17 1555, and ( + )-3-PPPl, because filtration of the soluble extracts from tissues exposed to these drugs did not reverse their effects. Furthermore, in agreement with Waggoner et al. (1980), we observed that 7-OH-DPAT, even at 0.1 mM, exerted no direct effect on TH activity. There- fore, TH change induced by 7-OH-DPAT [and LY 171555 or (+)-3-PPP] probably resulted from alterations in the intrinsic properties of the enzyme triggered by the stimulation of DA (auto)receptors lo-

J . Neurorhem.. Vol. 46, No. 1. 1986

TH CONTROL BY PRESYNAPTIC D A AUTORECEPTORS 19

0.1

5 0.6 E Y)

5 g 0.5

;

a

9 P I

E - n 0.3 n 0

I 0.2

0.1

aa

,E 0.7 In s g 0.6

E L

0.6 E E -

0.4 0

I n a3

0.2

0.1

0

-

-

-

-

- -

-

-

0,

T 1

- - Veratrldlne - - + + (-)-Sulpiride - t - + - +

a-Amphetrmlne - - - - + + FIG. 5. Effects of (-)-sulpiride on TH activity from striatal slices incubated with veratridine or d-amphetamine. Striatal slices were incubated for 15 min at 37°C in normal medium (4.8 mM K+) containing 10 pM veratridine, 10 pM (-)-sulpiride, andlor 5 pM d-amphetamine as indicated. Tissues were homogenized in 20 mM Tris-HCI, pH 7.0, containing 2 mM p-mercaptoethanol and centrifuged, and TH activity was measured in each supernatant. Data are mean f SEM values of TH activity (in nmol of [3H]DOPA/mg of protein/l5 min) in six separate samples. 'p < 0.05 compared with TH activity from control slices (no drug). "p < 0.05 compared with TH activity from corresponding tissues incubated in the absence of (-)-sulpiride (i.e., in the presence of veratridine or d-amphetamine alone).

cated on dopaminergic terminals themselves. As expected from such an interpretation, we observed that DA antagonists (haloperidol, sulpiride, and clozapine) prevented the inhibitory effect of 7-OH- DPAT on TH activity in isolated striatal nerve endings. Furthermore, (-)-sulpiride was -100 times more potent than (+)-sulpiride in this respect, in agreement with the known respective activities of these two enantiomers on DA receptors (Arbilla and Langer, 1981 ; Dubocovich and Weiner, 1981). Finally, 7-OH-DPAT and ( - )-sulpiride affected neither TH activity in hippocampal noradrenergic terminals nor tryptophan hydroxylase activity in cortical serotoninergic terminals, further confirming that the drug effect on striatal TH probably involves specific receptors on doparninergic terminals.

Previous studies on the negative control of DA release via presynaptic DA autoreceptors have led to the conclusion that these receptors exhibit pharmacological properties typical of D, binding sites

z 4 0 60 4.8 60

FIG. 6. Influence of CaZf on (-)-sulpiride-induced increase in TH activity in striatal slices depolarized by an excess of K f . Striatal slices were incubated for 15 rnin at 37°C in normal (4.8 mM K+) or K+-enriched (60 rnM) medium containing 1.3 mM or no Ca2+. In the latter case, 0.1 mM EGTA was added. For each incubating condition there were two groups: one with no drug (open bars) and the other with 10 pM (-)-sulpiride (stippled bars) added to the incubating medium. Tissues were homogenized in 20 mM Tris-HCI. pH 7.0, containing 2 mM pmercaptoethanol and centrifuged. TH activity was assayed in each supernatant and expressed in nmol of 13H]DOPNmg of proteinll5 min. Data are mean ? SEM values of six independent determinations. 'p < 0.05 compared with TH activity from control slices (no drug) incubated in complete normal medium (4.8 mM K+, 1.3 mM Ca'+). "p < 0.05 compared with TH activity from tissues depolarized in the presence of Ca2+ (60 mM K', 1.3 mM Ca2+) but in the absence of (-)-sulpiride.

(Stoof et al., 1982; Lehmann et al., 1983; Starke et al., 1983). Selective D, agonists have been used in the present study, but all were not active with respect to TH modulation: A significant decrease in TH activity was found in the soluble extracts of stnatal slices exposed to LY 171555 (see also Stoof and Kebabian, 1984), but no change was detected when tissues were exposed to other D, agonists such as lisuride, bromocnptine, or lergotrile (see also Kebabian and Kebabian, 1978). Although these observations may suggest that the autoreceptor controlling TH activity is not strictly of the D, type, any firm conclusion in this respect will require further investigations with complete dose-response curves using selective D, agonists and antagonists. In contrast to the results in tissues incubated

under nondepolarizing conditions, DA agonists did not affect TH activity in striatal slices incubated in K+-enriched medium. At least two hypotheses could be proposed to explain this difference. (a) Be- cause K +-induced depolarization triggers the acti-

J . Nerirocher.. Vol. 46, No. 1. 1986

20 S . EL MESTIKAWY ET A L .

TABLE 3 . Lack of effect of DA antagonists on TH artitity from striatal slices of reserpine-treated rats

TH activity (nmol ['HjDOPNmg proteidl5 min)

Addition [K'] 4.8 mM [ K - ] 60 mM

None 0.547 -t 0.053 0.942 z 0.080" ( - )-Sulpiride (10 pA4) 0.539 2 0.047 0.951 % 0.071 Haloperidol ( I pM) 0.566 2 0.043 0.941 ? 0.063 Methiothepin (10 pM) 0.559 2 0.062 0.911 z 0.098

Reserpine ( I0 mgkg intraperitoneally) was administered 24 h before the animals were killed. Striatal slices were incubated for I5 min at 37°C in normal (4.8 mM K') or K'-enriched (60 mM) medium containing ( - )-sulpiride, haloperidol. methiothepin. or no drug. Tissues were homogenized in 20 mM Tris-HCI, pH 7.0. containing 2 mM B-mercaptoethanol and centrifuged. TH activity was assayed in each supernatant and expressed in nmol of ['H]DOPA/mg of proteidl5 min. Data are mean 2 SEM values of six independent determinations.

a p < 0.05 compared with TH activity from tissues incubated in normal medium (4.8 mM K * ).

vation of TH in dopaminergic terminals, the intrinsic properties of the enzyme might be altered in such a way that it was not longer susceptible to the regulatory mechanism triggered by presynaptic DA autoreceptors. (b) Because K + -induced depolarization produces a massive efflux of DA (Arbilla and Langer, 1980), the presynaptic DA autoreceptors were already fully stimulated by the endogenous neurotransmitter, and no further effect could be induced by an exogenous agonist. According to this view, full negative control of TH should be oper- ating via presynaptic DA autoreceptors (in spite of the enzyme activation) in dopaminergic terminals incubated with 60 mM K + . Therefore, it should be possible to enhance TH activity further by pre- venting the stimulatory effect of endogenous DA on presynaptic autoreceptors. The present findings

TABLE 4. Effects of 7-OH-DPAT or ( - )-srilpiride on TH activity from striatal slices incuhoted in the uhsenc-e

or the presence of dibutyql cyclic AMP

TH activity (nmol ['H]DOPA/mg proteidl5 min)

Addition Dibutyryl

None cyclic AMP ~ _ _ _ _

None 0.342 2 0.042 0.7% 2 0.058

(-)-Sulpiride (1 pM) 0.338 f 0.031 0.786 f 0.059

Striatal slices were incubated for 15 min at 37°C in the absence or the presence of 2.5 mM dibutyryl cyclic AMP and 7-OH- DPAT, ( - )-sulpiride, or no drug. Tissues were homogenized in 20 mM Tris-HCI. pH 7.0, containing 2 mM 0-mercaptoethanol and centrifuged. TH activity was measured in each supernatant and expressed in nmol of [3HlDOPA/mg of proteidl5 min. Data are mean -c SEM values of six independent determinations.

a p < 0.05 compared with TH activity from striatal slices incubated in the absence of 7-OH-DPAT and ( - )-sulpiride.

7-OH-DPAT(I a) 0.251 f 0.021" 0.771 f 0.047

with ( - )-sulpiride and other DA antagonists were in complete agreement with this interpretation. In- deed, we observed that DA antagonists. which exerted no effect on the enzyme in control tissues, increased TH activity in striatal slices depolarized by 60 mM K + . As expected from the involvement of specific DA receptors, we noted that the active enantiomer ( - )-sulpiride was much more effective than (+)-sulpiride in enhancing TH activation in depolarized striatal slices. Furthermore, ( - )-3-PPP also exerted a stimulatory effect on TH activity in depolarized tissues. and several authors (Arbilla and Langer, 1984; Mulder et al.. 1985) have recently provided evidence for ( - )-3-PPP acting as an antagonist of presynaptic DA autoreceptors. Finally, comparison of the respective activities of chlorpromazine and of its sulfoxide metabolite confirmed the implication of DA receptors, because an increase in TH activation due to depolarization was noted only with chlorpromazine, which is at least 100 times more active than its metabolite as a DA antagonist in the rat striatum (Miller and Iversen, 1974).

The present experiments with ( - )-sulpiride and other antagonists demonstrated therefore that TH activity could be controlled by presynaptic autoreceptors in depolarized dopaminergic terminals. However, depolarization was not necessary per se, because a significant activation of TH was also induced by ( - )-sulpiride in striatal slices exposed to the DA-releasing drug d-amphetamine. which has been shown by Schwarz et al. (1980) and our present work to enhance by itself DA synthesis but not to activate TH. Therefore, it could bc proposed that K + -induced depolarization made TH susccp- tible to activation by ( - )-sulpiride (and other antagonists) only becausc it induced DA rclcasc. In- deed, under conditions resulting in a markcd rcduc- tion of DA releasc from dcpolarizing ti\sucs, i.c.. the elimination of Ca? ' in the incuhating mcdium or in vivo trcatment with rescrpinc. ( - )-sulpiridc did not enhance TH activation in depolarized dopaminergic terminals. Taken together, all thcse observations strongly support the proposal that cn- dogenous DA relcased in thc synaptic clcft inhihits TH activity via the stimulation of functional prc- synaptic autoreceptors on striatal dopaminergic terminals.

Studies on the regulatory mechanisms of TH in dopaminergic neurons have revealed that intrinsic properties of the enzyme can be altered by two dif- ferent phosphorylation processes. one involving the cyclic AMP-dependent protein kinase (Morgenroth et al., 1975) and the other Ca?+-dependent protein kinases (El Mestikawy et al., 1983, 1985: Iuvone, 1984). Previous investigations by Roth and co- workers (Billingsley and Roth, 1982; Roth, 1984; Wolf and Roth, 1985) have led to the proposal that TH control by presynaptic DA autoreceptors might

1. Neurorhem.. Vol . 46, No. I . 1986

TH CONTROL BY PRESYNAPTZC DA AUTORECEPTORS 21

concern the Ca2+-dependent phosphorylation process via the carboxymethylation of calmodulin. However, we observed that TH regulation by presynaptic autoreceptors occurred when the enzyme was fully activated by Ca*+-dependent phosphorylation (in depolarized tissues; see El Mestikawy et al., 1983, 19851, but not when TH was activated by the cyclic AMP-dependent process (in slices incubated with dibutyryl cyclic AMP; Table 4). The lack of effect of (-)-sulpiride or 7-OH-DPAT on TH activity in striatal slices exposed to dibutyryl cyclic AMP did not result from some insensitivity of the activated enzyme to any regulatory process, because previous studies have indicated that TH in dibutyryl cyclic AMP-exposed tissues can be further activated by depolarization (El Mestikawy et al., 1983, 1985). Instead, it could be proposed that ( -)-sulpiride and 7-OH-DPAT were inactive because they normally control a process that is fully achieved by dibutyryl cyclic AMP, i.e., the cyclic AMP-dependent phosphorylation of TH. Prelirni- nary data support the suggestion that the reduction in TH activity in dopaminergic terminals exposed to some DA agonists (notably 7-OH-DPAT) resulted from a decreased proportion of the TH form phos- phorylated by the cyclic AMP-dependent protein kinase (El Mestikawy and Hamon, 1985). Whether the presynaptic DA autoreceptor is, like the D, receptor (Enjalbert and Bockaert, 1983; Berry-Kravis et al., 1984; Stoof and Kebabian, 1984), negatively coupled to adenylate cyclase is therefore an unan- swered question.

Acknowledgment: This research has been supported b y g r a n t s f r o m I N S E R M a n d l e s U s i n e s Chimiques RhBne-Poulenc. We are grateful to the following compa- nies: Delagrange, Eli Lilly & Co., Hoffmann-La Roche, Janssen , RhBne-Poulenc, Sandoz, Schering, and Syn- thelabo (Dr. S. Z. Langer), and t o Dr. E. Jover (INSERM U 172, Marse i l le , F r a n c e ) a n d Dr. A. S. H o r n (Gro- ningen, T h e Nether lands) f o r the i r g e n e r o u s gif ts of drugs.

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