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Central regulation of adrenal tyrosine hydroxylase: Interaction between dopamine and GABA systems
NeuropharmacologyVol. 28, No. 5, pp. 521-527, Printed
in Great
Britain.
1989 Copyright
All rights reserved
0028-3908/89 53.00 + 0.00 @$I1989 Pergamon Press plc
CENTRAL REGULATION OF ADRENAL TYROSINE HYDROXYLASE: INTERACTION BETWEEN DOPAMINE AND GABA SYSTEMS R. GABOR,’ S. REGUNATHAN*and T. L. SOURKES’ ‘Departments of Biochemistry and Psychiatry, Faculty of Medicine, McGill University, Montreal, and 2Douglas Hospital Research Centre, Verdun, Quebec, Canada (Accepted 20 October 1988)
Summary-It has been previously demonstrated that nigrostriatal dopaminergic fibres participate in the neural regulation of the activity of adrenal tyrosine hydroxylase, specifically in its induction. To determine whether activation or inhibition of these fibres is responsible for this induction, the role of presynaptic dopamine receptors was investigated. Apomorphine (0.2 mg/kg), ( + )3-PPP (10 mg/kg) and BHT 920 (1-3 mg/kg), drugs that are reported to bind to presynaptic dopamine receptors and thereby inhibit the release of that neurotransmitter, caused significant increases in the activity of the enzyme. As a central GABA (y-aminobutyric acid) system is believed to exert inhibitory control over the release of dopamine, GABA agonists were also tested for their effects. Muscimol (3 mg/kg), y-hydroxybutyrate (500 mg/kg) and HA-966 (150 mg/kg) produced significant induction of the adrenal enzyme; this induction was not blocked by dopamine postsynaptic receptor antagonists. After intraventricular administration (5 pg/rat) in normal animals, HA-966 produced significant induction of tyrosine hydroxylase. Its systemic administration did not induce the enzyme in animals with the adrenal denervatedwhen administered together at submaximal doses, HA-966 and BHT 920 produced an additive effect in the induction of adrenal tyrosine hydroxylase. Key
words-adrenal hyhroxylase.
medulla,
dopamine,
GABA,
Studies of the neural regulation of adrenal enzymes have helped elucidate the central pathways involved in stress responses. Transneuronal induction under the influence of stressors plays an important role in this regulation (Thoenen, Mueller and Axelrod, 1969; Ciaranello, 1980). The changes elicited by stressors can be mimicked by using various drugs to manipulate central neurotransmitter systems such as dopaminergic, serotonergic and cholinergic systems (Lewander, Joh and Reis, 1977; Sourkes, 1985; 1987). Earlier work from this laboratory demonstrated that a supraspinal dopaminergic system (Gagner, Gauthier and Sourkes, 1983), located in the striatal region of the rat (Ekker and Sourkes, 1985), plays an important role in the regulation of adrenal tyrosine hydroxylase (Quik and Sourkes, 1976; Sourkes, 1987). Thus, dopamine (DA) agonists induce adrenal tyrosine hydroxylase and antagonists of dopamine block the induction. At the time these findings were established, it was concluded that the postsynaptic activation of a central dopaminergic system leads to the induction of adrenal tyrosine hydroxylase (Quik and Sourkes, 1977). However, more recent neurochemical and neurophysiological studies of the dopamine system in the basal ganglia have indicated that the actions of dopaminergic drugs may involve rather more complex mechanisms. For example, presynaptic receptors for dopamine (autoreceptors) have been identified and this has led to the discovery that dopamine agonists can bind to these receptors also, NP 28,SF
521
nigrostriatal
tract,
strionigral
fibres, tyrosine
and consequently inhibit the release of dopamine (Aghajanian and Bunney, 1977; StPhle and Ungerstedt, 1984). The strionigral feedback loop has been implicated in the control of firing in the nigrostriatum and a GABA system is involved in this (Starr, Summerhayes and Kilpatrick, 1983; Scheel-Kriiger, 1986). GABAergic neurons in both the substantia nigra and the striatum provide additional control over the nigrostriatal output (Oertel and Mugnaini, 1984; Ribak, Vaughn and Roberts, 1979). Thus, the presumed regulation of adrenal tyrosine hydroxylase by a central dopaminergic system may also involve central GABA systems. In the present work the main objectives were to assess (1) the role of presynaptic DA receptors; (2) the role of central GABAergic systems; and (3) the interaction between these systems in the transneuronal induction of adrenal tyrosine hydroxylase. METHODS AnimaIs
Male Sprague-Dawley rats weighing 200-250 g were used. They were obtained from the breeding farms of Charles River Canada Incorporated (St Constant, Quebec). They were kept 4 or 5 to a cage in an animal room with a light-dark cycle of 12 hr and with a thermostatically controlled temperature of 22°C. In experiments involving the adminstration of apomorphine, the animals were placed in individual
522
R. GABOR et al.
wire cages. There was free access to tap water and Purina Checkers. Control animals received vehicle by the same route as the experimental group and also received the same volume and number of injections. The rats were handled frequently before use in experiments in order to accustom them to the experimenters. Experiments were not begun until at least the morning after receipt of the animals. At the end of an experiment, the rats were sacrificed by decapitation. Surgery Intracerebroventricular injection of drugs. For these experiments, rats weighing between 200 and 210 g, were anesthetized with chloral hydrate, 300 mg/kg (ip.) (USP, Fisher Scientific Company, Montreal, Canada) and positioned in the stereotaxic instrument. With the skull horizontal, the injections were given at the point 1.0 mm posterior to bregma, 1.5 mm lateral to midline and 3.5 mm vertical (Paxinos and Watson, 1982). The ventricular sitetwas confirmed by injection of methylene blue. The drugs were given in a volume of 10 ~1 delivered over a period of 2-min from a Hamilton microsyringe. Animals were sacrificed 24 hr after the administration of drugs. Splanchnicotomy. Section of the left splanchnic nerve was performed in rats under chloral hydrateinduced anesthesia, 300 mg/kg (ip.). The animals for this part of the work weighed between 200 and 220 g at the time of surgery. With a dissecting microscope, the tissue surrounding the adrenal gland was completely dissected, except for protection of the vascular supply to the gland. The mean weights of the adrenals on the denervated and intact sides were not significantly different. To verify the efficacy of the operation, in a few rats the site of the transsection was electrically stimulated and heart rate and blood pressure were measured. With the technique used, no increase in heart rate or blood pressure was detected. Injections were begun on the fifth post-operative day in order to allow for sufficient recovery from surgical stress. Drugs
The following drugs were purchased: sodium y-hydroxybutyrate and muscimol, from Sigma Chemical Company, St Louis, Missouri; apomorphine HCl from F. E. Cornell and Co., Montreal, Quebec; and R( +)-(3_hydroxyphenyl)N-n -propylpiperidine [( + )3-PPP] from Research Biochemicals Inc., Wayland, Massachusetts. The following drugs were gifts and are gratefully lo-hydroxy-3-amino-pyrrolidone-2 acknowledged: (HA-966) from Dr E. L. Noach, U. of Leiden and from Organon, Oss, The Netherlands; haloperidol from McNeil Laboratories, Don Mills, Ontario; R( + )-8-chloro-2,3.4,5-tetrahydro-3-methyl-5-phenyl1H-3-benzazepine-7-OH (SCH 23390) from Schering-Plough Co., Bloomfield, New Jersey; racemic sulpiride from Delagrange International, Paris,
France; d-sulpiride and I-sulpiride from Ravizza S.P.A., Milan, Italy; and 6-allyl-2-amino-5,6, 7,8-tetrahydro-4H-thiazolo-(4,5-d)azepine dihydrochloride (B-HT 920; BHT 920) from Boehringer (Ingelheim) Canada, Ltd, Burlington, Ontario. Most of the drugs were dissolved in saline, but y-hydroxybutrate was dissolved in water and titrated to approximately pH 7.2 with a few drops of 1N HCl. Haloperidol, sulpiride and HA-966 were each dissolved in a few drops of glacial acetic acid, diluted with deionized water and titrated to pH 6.3; SCH 23390 was dissolved in a 25% solution of propylene glycol. All drugs were injected in a volume of 0.5 ml. Control animals received the vehicle in the same volume as the drug and with the same number of injections. Assay of tyrosine hydroxylase
The rats were killed by decapitation. The adrenals were quickly removed, cooled on ice, freed from capsular tissue and weighed. Whole adrenals were used for determination of the activity of adrenal tyrosine hydroxylase. This was assayed according to the method of Nagatsu, Levitt and Udenfriend (1964), as modified by Gauthier, Gagner and Sourkes (1979). Statistical analysis
Values in all Tables are expressed as means f SE. Student’s t-test was used for the comparison of paired means (Snedecor, 1956). In most experiments a two-tailed t-test was performed. However, when the direction of effect of a particular treatment was already established and the effect of a further drug on that vector was to be determined, a one-tailed t-test was often applied. For two-way experiments (drug A x drug B) and three-way experiments (drug A x drug B x replications), the analysis of variance (ANOVA) was used (Snedecor, 1956). RESULTS
Effect of apomorphine
Initially it was necessary to determine the relationship of the activity of adrenal tyrosine hydroxylase to the dose of apomorphine. From the results in Figure 1, it is evident that tyrosine hydroxylase was induced over a wide range of doses of apomorphine but that as little as 0.1-0.2mg/kg was effective. In the next experiment, the ability of two postsynaptic dopamine-receptor blockers, namely SCH23390 and I-sulpiride (Iorio, Barnett, Leitz, Houser and Korduba, 1983; Honda, Satoh, Shimonura, Satoh, Noguchi, Uchida and Kato, 1977), to abolish the induction caused by apomorphine was tested. In this case, apomorphine in a dose of 3 mg/kg, was injected 30 min after the administration of SCH23390 (3 mg/kg i.p., twice daily) or I-sulpiride (40 mg/kg S.C.once daily) to separate groups of rats. In the doses used, neither of these antagonists alone
Control
of
523
adrenal tyrosine
received apomorphine, SCH23390 caused decreases in the mean level of activity of adrenal tyrosine hydroxylase, but sulpiride did not do so (Table 1). Efect
of dopamine autoreceptor agonists
In order to test further the possibility of an action at autoreceptors, drugs that are reported to be specific for dopamine autoreceptors were used in the next set of experiments. The first compound tried was 3-(3hydroxyphenyl)-N-n-propylpiperidine [( + )3-PPP], a compound that binds presynaptic DA receptors and inhibits the release of dopamine (Hjorth, Carlsson, Wikstrom, Lindberg, Sanchez, Hacksell, Arvidsson, 0 0.1 0:2 0:4” 018 ” ,:s Svensson and Nilsson, 1981). It was administered to APOMORPHINE (mg/kgl rats in a dose of 10 mg/kg (s.c.), once daily for 3 days. Fig. 1. Effect of apomorphine on the activity of adrenal As shown in Table 2, this treatment elicited a tyrosine hydroxylase. Values are mean + SE of indicated significant inductive effect on the activity of adrenal number of animals in the brackets. Apomorphine was tyrosine hydroxylase. The intracerebroventricular dissolved in saline and the animals received the indicated administration of (+)3-PPP in the amount of amount in a volume of 0.5 ml (s.c.), four times a day for 3 days. Animals were killed on the fourth day. aP 0.05 in both cases). agonist tested was BHT 920. This substance is more When the drugs were tested in rats that had also Table
I. Effect of SCH 23390 and sulpiride on apomorphine-elicited increase in the activity of adrenal tyrosine hydroxylase Activity of tyrosine hydroxylase
Treatment Apomorphine Apomorphine + SCH 23390 Apomorphine Apomorphine + sulpiride
55.8 f 41.1 f 58.5 f 50.3 f
4.75 (8) 2.84 (9)’ 4.41 (8) 4.61 (8)
The activity of tyrosine hydroxylase is expressed as nmol dihydroxyphenylalanine (DOPA) formed per hr per pair of adrenals. Values are mean f SE for the indicated number of animals. Drugs were administered as follows: SCH23390: 3 mg/kp (i.p.) twice daily; sulpiride; 40 mgjkg (s.c.) once daily; apomorphine: 3 mg/kg (s.c.) 30 mitt following the injections of antagonists. The animals were treated for 3 days and killed on the fourth day. ‘P < 0.05 compared to apomorphine-treated rats. Table 2. Effect of ( + )3-PPP and GABAergic drugs on the activity of adrenal tvrosine hvdroxvlase Experiment A B C D E F
Treatment Control ( + )3-PPP (S.C.) Control ( + )3-PPP (i.c.v.) Control Muscimol (s.c.) Control GOBA (i.p.) Control HA-966 (i.p.) Control HA-966 (i.c.v.)
Activity of tyrosine hydroxylase 33.4 k 42.9 + 25.7 f 32.7 f 31.6 f 59.7 + 33.1: 41.2+ 24.8: 35.1 + 39.2 I 51.5 f
2.48 (9) 2.34’ (IO) I .98 (7) I .74’ (9) 2.05 (23) 1.97**(25) 1.4(14) 1.31**(16) 1.12(15) 1.08**(16) 3.25 (8) 3.76*** (6)
Enzyme activity is expressed as in Table I. The drugs were injected as follows: ( + )3-PPP: IOmg/kg once daily for 3 days; for the intracerebroventricular i.c.v.) experiment with (+ )3-PPP, one injection of 2 pg/rat was given. y-Hydroxybutyrate (GOBA): 0.5mg/kg once daily for 3 days; HA-966: I SOmg/kg once daily for I day; muscimol: 3 mg/kg twice a day for 3 days; for the inttacerebroventricular (i.c.v) experiment with HA-966, one injection of 5 pg per rat was given. All animals were killed on the fourth day. Values are mean + SE of indicated number of animals. Probabilities for comparison with saline controls are: *P < 0.05; **P < 0.01; ***p < 0.005.
524
R. GABORet al. Table 3. Effect of HA-966 on denervated adrenal tyrosine hvdroxvlase Activity of tyrosine hydroxylase” Treatment Hemi-splanchnicotomy Hemi-splanchnicotomy
+ saline + HA-966
Innervated
Denervated
19.9 + 3.26 (7) 40.9 f 2.88** (9)
21 .Ok 4.32 (4) 16.2 + 3.26’ (7)
-
‘Per single gland. The HA-966 was given in a dose of I50 mg/kg once daily for 3 days by the intraperitoneal route and animals were killed on the fourth day. Two experiments were carried out and the results were subjected to ANOVA. This yielded a mean square for error of 74.5234 (I9 d.f.) which was used for estimating the SE of means of various sizes and of differences between the means. *Not significantly different from the means for control adrenals (P z 0.05). **Significantly different from the other means (P < 0.005).
specific than ( + )3-PPP for presynaptic receptors and is devoid of activity on postsynaptic receptors (Anden, Nilsson, Ros and Thornstrom, 1983). It was injected in doses of 1, 2 and 3 mg/kg (s.c.) once daily for 3 days. The activity of adrenal tyrosine hydroxylase increased in a dose-dependent fashion as a result of this treatment (Fig. 2). Effect of GABAergic drugs
The influence of a GABA system on the induction of the adrenal enzyme was studied by measuring the activity of adrenal tyrosine hydroxylase after the injection of various GABAergic agents. The first drug tested was muscimol, a GABAA receptor agonist. This, when administered to rats in a dose of 3 mg/kg (s.c.) produced a very significant increase in the activity of adrenal tyrosine hydroxylase (Table 2). Sodium y -hydroxybutyrate and HA-966, two analogues of GABA, which temporarily prevent the release of dopamine from nigrostriatal fibres (Walters, Roth and Aghajanian, 1973; Hillen and Noach, 1971), were also tested for their effects on the activity of the adrenal enzyme. As shown in Table 2, y -hydroxybutyrate given once daily for 3 days by the intraperitoneal route caused a significant induction of adrenal tyrosine hydroxylase. In the case of HA-966, a single dose, given intraperitoneally, caused a mean increase of 42% in the activity of adrenal tyrosine
hydroxylase, as measured 96 hr later. Because of the pronounced effect of HA-966, this compound was tested by single intracerebroventricular injection. in the amount of 5 pgg/rat, the animals being killed on the fourth day (96 hr later). Again, as seen in Table 2, a very significant induction of adrenal tyrosine hydroxylase was observed. In order to confirm the central action of the GABAergic drugs, HA-966 was tested in rats that had one adrenal gland denervated some days before the treatment with drugs began. In these animals, the nerve input to the left adrenal gland was eliminated by splanchnicotomy; the innervation of the right gland remained intact. The results from such an experiment are shown in Table 3. The HA-966 caused a large increase in the activity of tyrosine hydroxylase of the innervated gland, but was ineffective on the denervated side. Interaction between GABAergic drugs
The ability of dopamine postsynaptic-receptor blockers to abolish the induction of adrenal tyrosine hydroxylase by GABA analogues was tested by the use of three dopamine antagonists, namely haloperidol, I-sulpiride and SCH23390. These drugs were injected 30min prior to the administration of the GABAmimetics. None blocked the induction of adre-
Table 4. Effect of DA blocking agents on the induction of adrenal tyrosine hydroxylase, caused bv GABAernic druns Exoeriment A
B
C
Treatment
Activity of tyrosine hydroxylase
Sulpiride GOBA GOBA + Sulpiride HA-966 HA-966 + Sulpiride
46.1 *3.16(S) 45.3 f 3.16(S)* 34.2 f 1.86 (7) 32.4 f 1.74 (8)’
SCH 23390 HA-966 HA-966 + SCH 23390
26.8 k 2.4 (7) 28.5 f 2.24 (8)’
Haloperidol Muscimol Muscimol +
and dopaminergic
53.5 i 6.92 (5) 68.4 i 5.49 (6)’
Halooeridol Drugs were administered as follows: GOBA: 0.5 g/kg (i.p.) once daily for 3 days; HA-966: I50 mg/kg (i.p.) for I day; muscimol: 3 mg/kg (s.c.) twice daily for 3 days: sulpiride: 40 mg/kg (s.c.) once daily 30 min prior to GABA drugs; SCH 23390: 3 mg/kg, (i.p.) twice daily, 30 min prior to HA-966; haloperidol; 5 mg/kg, (i.p.) once daily, 60 min prior to muscimol. Animals were killed on the morning after the last injection. Values are mean f SE of indicated number of animals. *P > 0.05 for comparison with corresponding control mean (no blocking agent).
Control of adrenal tyrosine Table 5. Effect of HA-966 and BHT 920 on the activity tyrosine hydroxylase Treatment Control HA-966 BHT 920 HA-966 + BHT 920
Activity
of tyrosine
25.1 37.2 36.0 49.6
of adrenal
hydroxylase
f 2.59 (3) (100%) + 1.47” (4) (149) : I .65* (4) (143) _+ 3.85” (5) (198)
The drugs were injected as follows: HA-966: 100 mg/kg, (Lp.) once daily for 3 days; BHT 920: 1 mg/kg, (s.c.) once daily for 3 days; for animals receiving both drugs, the same amount of drug was given 30 min apart. All animals were sacrificed on the fourth day. Values are mean f SE of indicated number of rats. Probabilities for comparison with saline controls are: *P < 0.025; l*P < 0.005.
nal tyrosine hydroxylase produced by the GABAergic substances (Table 4). If GABAergic drugs and dopamine autoreceptor agonists both act by inhibiting the release of dopamine, then the administration of these drugs together, at submaximal doses, should produce an additive response. This was tested by giving rats HA-966 (100 mg/kg) and BHT 920 (1 mg/kg), 30 min apart, daily for 3 days. The results in Table 5 show that the mean increase of 98%, obtained when the drugs were given together, was precisely an additive effect. DISCUSSION
Earlier work from this laboratory established the involvement of central dopaminergic systems in the induction of adrenal tyrosine hydroxylase (Quik and Sourkes, 1976). The large doses of apomorphine (3-lOmg/kg), used in earlier work, caused stimulation of both pre- and postsynaptic receptors and thus were not discriminatory. From the present work (Fig. l), it was evident that doses as small as 0.14.2mg/kg of apomorphine were effective in inducing adrenal tyrosine hydroxylase and that larger doses produced no further effect. In the small doseinteracts mainly with prerange, apomorphine synaptic dopamine receptors and thereby inhibits the release of dopamine (StPhle and Ungerstedt, 1984; Zetterstriim and Ungerstedt, 1984). The involvement of presynaptic receptors was further substantiated by the use of postsynaptic dopamine-receptor blockers. The two drugs used, namely SCH23390 and lsulpiride, produced different effects. The failure of sulpiride LO affect significantly the action of apomorphine, points to the role of a presynaptic component in the induction of the enzyme. The inhibitory action of SCH23390 in this test seemed to illustrate the fact that this compound can interact with both D, and D, receptor types, including Dz autoreceptors of the type found predominantly at dopaminergic nerve endings (Creese, 1987; Goldstein, Litwin, Sutton and Malick, 1987; Morelli and Di Chiara, 1985; Plantje, Daus, Hansen and Stoof, 1984). Because the use of postsynaptic receptor blockers did not provide conclusive evidence for the involvement of presynaptic receptors, two drugs that
525
are reported to be specific for autoreceptors were used. Both drugs, namely ( + )3-PPP and BHT-920, proved to be effective in inducing adrenal tyrosine hydroxylase (Table 2; Fig. 2). The effect of BHT-920 was dose-dependent, as analyzed by multiple regression analysis (Fig. 2). This substance is said to be more specific than (+)3-PPP for autoreceptors and is devoid of activity at postsynaptic receptors (Anden, Nilsson, Ros and Thornstrom, 1983). These experiments, then, demonstrate clearly that the inhibition of the release of dopamine by stimulating autoreceptors results in the induction of adrenal tyrosine hydroxylase. The site of action of these drugs could be at the level of the neural loop formed by the strionigral and nigrostriatal tracts. A GABAergic system is known to provide feedback from the striatum to the substantia nigra and the activation of this system prevents nigrostriatal neurons from releasing dopamine (Walters et al., 1973; Hillen and Noach, 1971). Various GABA agonists, at least one of them (HA-966) now shown to act centrally, produced significant induction of adrenal enzyme in a manner similar to that of the dopamine autoreceptor agonists (Table 2). Because dopamine postsynaptic receptorblockers did not abolish the induction produced by GABA drugs (Table 4), postsynaptic stimulation is probably not involved in this type of induction. As mentioned earlier, if GABAmimetic drugs modulate the dopaminergic system, then drugs acting at the autoreceptors should be able to affect the GABAmediated induction. As there is no dopamine autoreceptor-antagonist available that can block specifically the presynaptic receptors, it was decided to follow a different approach: if GABAergic drugs and dopamine autoreceptor agonists both act by inhibiting the release of dopamine, then the adminis-
,
I
0
2
1 BHT
920
I
3
(mg/kgl
Fig. 2. Effect of BHT 920 on the activity of adrenal tyrosine hydroxylase. Values are mean f SE of 5 animals. The BHT 920 was dissolved in saline and the indicated amount was injected once daily in a volume of 0.5 ml (s.c.) for 3 days. Animals were killed on the fourth day. The slope (b + SE = 11.69 f 2.4) is significantly different from zero (P < 0.001, 18 degrees of freedom), as analyzed by multiple regression.
R. GABOR etal.
526
tration of such drugs together at submaximal doses should produce an additive response. The results from such an experiment (Table 5) show precisely an
additive effect when HA-966 and BHT 920 were given together. These observations provide conclusive evidence that stimulation of either a central GABA system or a dopamine autoreceptor can produce similar responses. Hitherto, it has been established that the central dopaminergic and cholinergic systems play an excitatory role, whereas (5hydroxytryptamine, 5-HT) contributes inhibitorily to the induction of adrenal tyrosine hydroxylase (Sourkes, 1985; Lewander et al., 1977). The present work has revealed a central GABAergic pathway which exerts a net excitatory influence on the induction of this enzyme. The similarity of inductive effects of small amounts of apomorphine dopamine-autoreceptor agonists and analogues of GABA, combined with the fact that GABA inhibits the nigrostriatal DA system, now suggests that the DA-elicited increase in adrenal tyrosine hydroxylase is a presynaptically mediated phenomenon. The fact that a GABA agonist and a DA autoreceptor agonist had an additive effect (Table 5) points to the striatonigral GABAergic feedback pathway as of importance in the control of the nigrostriatal dopaminergic system. The role of non-dopaminergic pathways in the regulation of the activity of adrenal tyrosine hydroxylase is demonstrated by the failure of dopamine antagonists to block the induction caused by analogues of GABA (Table 4). One such site of action of GABA has been discussed above. However, the presence of GABAergic neurons in the raphe nuclei, where they exert inhibitory control over the serotonergic cells therein (Nishikawa and Scatton, 1985; Belin, Nanopoulos, Didier, Aghera, Steinbusch, Verkofstad, Maitre and Peyot, 1983), opens up another possible site of action of GABA drugs in inducing the adrenal enzyme. Acknowledgements-This
work has been supported by a
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(+)-3-PPP and (-)-3-PPP by recording of yawning behavior in rats. Eur. J. Pharmac. 98: 307-310. Starr M. S., Summerhayes M. and Kilpatrick I. C. (1983) Interactions between dopamine and y-aminobutyrate in the substantia nigra; imnlications for the striatoniaral output hypothesis. Neurowience 8: 547-559. Thoenen H., Mueller R. A. and Axelrod J. (1969) Transsynaptic induction of adrenal tyrosine hydroxylase. J. Pharmac. exp. Ther. 169: 249-254. Walters J. R., Roth R. H. and Aghajanian G. K. (1973) Dopaminergic neurons; similar biochemical and histochemical effects of y-hydroxybutyrate and acute lesions of the nigrostriatal pathway. J. Pharmac. exp. Ther. 186: 63&639. Zetterstrom T. and Ungerstedt U. (1984) Effects of apomorphine on the in uiuo release of dopamine and its metabolites, studied by brain dialysis. Eur. J. Pharmnc. 97: 29-36.
in Great
Britain.
1989 Copyright
All rights reserved
0028-3908/89 53.00 + 0.00 @$I1989 Pergamon Press plc
CENTRAL REGULATION OF ADRENAL TYROSINE HYDROXYLASE: INTERACTION BETWEEN DOPAMINE AND GABA SYSTEMS R. GABOR,’ S. REGUNATHAN*and T. L. SOURKES’ ‘Departments of Biochemistry and Psychiatry, Faculty of Medicine, McGill University, Montreal, and 2Douglas Hospital Research Centre, Verdun, Quebec, Canada (Accepted 20 October 1988)
Summary-It has been previously demonstrated that nigrostriatal dopaminergic fibres participate in the neural regulation of the activity of adrenal tyrosine hydroxylase, specifically in its induction. To determine whether activation or inhibition of these fibres is responsible for this induction, the role of presynaptic dopamine receptors was investigated. Apomorphine (0.2 mg/kg), ( + )3-PPP (10 mg/kg) and BHT 920 (1-3 mg/kg), drugs that are reported to bind to presynaptic dopamine receptors and thereby inhibit the release of that neurotransmitter, caused significant increases in the activity of the enzyme. As a central GABA (y-aminobutyric acid) system is believed to exert inhibitory control over the release of dopamine, GABA agonists were also tested for their effects. Muscimol (3 mg/kg), y-hydroxybutyrate (500 mg/kg) and HA-966 (150 mg/kg) produced significant induction of the adrenal enzyme; this induction was not blocked by dopamine postsynaptic receptor antagonists. After intraventricular administration (5 pg/rat) in normal animals, HA-966 produced significant induction of tyrosine hydroxylase. Its systemic administration did not induce the enzyme in animals with the adrenal denervatedwhen administered together at submaximal doses, HA-966 and BHT 920 produced an additive effect in the induction of adrenal tyrosine hydroxylase. Key
words-adrenal hyhroxylase.
medulla,
dopamine,
GABA,
Studies of the neural regulation of adrenal enzymes have helped elucidate the central pathways involved in stress responses. Transneuronal induction under the influence of stressors plays an important role in this regulation (Thoenen, Mueller and Axelrod, 1969; Ciaranello, 1980). The changes elicited by stressors can be mimicked by using various drugs to manipulate central neurotransmitter systems such as dopaminergic, serotonergic and cholinergic systems (Lewander, Joh and Reis, 1977; Sourkes, 1985; 1987). Earlier work from this laboratory demonstrated that a supraspinal dopaminergic system (Gagner, Gauthier and Sourkes, 1983), located in the striatal region of the rat (Ekker and Sourkes, 1985), plays an important role in the regulation of adrenal tyrosine hydroxylase (Quik and Sourkes, 1976; Sourkes, 1987). Thus, dopamine (DA) agonists induce adrenal tyrosine hydroxylase and antagonists of dopamine block the induction. At the time these findings were established, it was concluded that the postsynaptic activation of a central dopaminergic system leads to the induction of adrenal tyrosine hydroxylase (Quik and Sourkes, 1977). However, more recent neurochemical and neurophysiological studies of the dopamine system in the basal ganglia have indicated that the actions of dopaminergic drugs may involve rather more complex mechanisms. For example, presynaptic receptors for dopamine (autoreceptors) have been identified and this has led to the discovery that dopamine agonists can bind to these receptors also, NP 28,SF
521
nigrostriatal
tract,
strionigral
fibres, tyrosine
and consequently inhibit the release of dopamine (Aghajanian and Bunney, 1977; StPhle and Ungerstedt, 1984). The strionigral feedback loop has been implicated in the control of firing in the nigrostriatum and a GABA system is involved in this (Starr, Summerhayes and Kilpatrick, 1983; Scheel-Kriiger, 1986). GABAergic neurons in both the substantia nigra and the striatum provide additional control over the nigrostriatal output (Oertel and Mugnaini, 1984; Ribak, Vaughn and Roberts, 1979). Thus, the presumed regulation of adrenal tyrosine hydroxylase by a central dopaminergic system may also involve central GABA systems. In the present work the main objectives were to assess (1) the role of presynaptic DA receptors; (2) the role of central GABAergic systems; and (3) the interaction between these systems in the transneuronal induction of adrenal tyrosine hydroxylase. METHODS AnimaIs
Male Sprague-Dawley rats weighing 200-250 g were used. They were obtained from the breeding farms of Charles River Canada Incorporated (St Constant, Quebec). They were kept 4 or 5 to a cage in an animal room with a light-dark cycle of 12 hr and with a thermostatically controlled temperature of 22°C. In experiments involving the adminstration of apomorphine, the animals were placed in individual
522
R. GABOR et al.
wire cages. There was free access to tap water and Purina Checkers. Control animals received vehicle by the same route as the experimental group and also received the same volume and number of injections. The rats were handled frequently before use in experiments in order to accustom them to the experimenters. Experiments were not begun until at least the morning after receipt of the animals. At the end of an experiment, the rats were sacrificed by decapitation. Surgery Intracerebroventricular injection of drugs. For these experiments, rats weighing between 200 and 210 g, were anesthetized with chloral hydrate, 300 mg/kg (ip.) (USP, Fisher Scientific Company, Montreal, Canada) and positioned in the stereotaxic instrument. With the skull horizontal, the injections were given at the point 1.0 mm posterior to bregma, 1.5 mm lateral to midline and 3.5 mm vertical (Paxinos and Watson, 1982). The ventricular sitetwas confirmed by injection of methylene blue. The drugs were given in a volume of 10 ~1 delivered over a period of 2-min from a Hamilton microsyringe. Animals were sacrificed 24 hr after the administration of drugs. Splanchnicotomy. Section of the left splanchnic nerve was performed in rats under chloral hydrateinduced anesthesia, 300 mg/kg (ip.). The animals for this part of the work weighed between 200 and 220 g at the time of surgery. With a dissecting microscope, the tissue surrounding the adrenal gland was completely dissected, except for protection of the vascular supply to the gland. The mean weights of the adrenals on the denervated and intact sides were not significantly different. To verify the efficacy of the operation, in a few rats the site of the transsection was electrically stimulated and heart rate and blood pressure were measured. With the technique used, no increase in heart rate or blood pressure was detected. Injections were begun on the fifth post-operative day in order to allow for sufficient recovery from surgical stress. Drugs
The following drugs were purchased: sodium y-hydroxybutyrate and muscimol, from Sigma Chemical Company, St Louis, Missouri; apomorphine HCl from F. E. Cornell and Co., Montreal, Quebec; and R( +)-(3_hydroxyphenyl)N-n -propylpiperidine [( + )3-PPP] from Research Biochemicals Inc., Wayland, Massachusetts. The following drugs were gifts and are gratefully lo-hydroxy-3-amino-pyrrolidone-2 acknowledged: (HA-966) from Dr E. L. Noach, U. of Leiden and from Organon, Oss, The Netherlands; haloperidol from McNeil Laboratories, Don Mills, Ontario; R( + )-8-chloro-2,3.4,5-tetrahydro-3-methyl-5-phenyl1H-3-benzazepine-7-OH (SCH 23390) from Schering-Plough Co., Bloomfield, New Jersey; racemic sulpiride from Delagrange International, Paris,
France; d-sulpiride and I-sulpiride from Ravizza S.P.A., Milan, Italy; and 6-allyl-2-amino-5,6, 7,8-tetrahydro-4H-thiazolo-(4,5-d)azepine dihydrochloride (B-HT 920; BHT 920) from Boehringer (Ingelheim) Canada, Ltd, Burlington, Ontario. Most of the drugs were dissolved in saline, but y-hydroxybutrate was dissolved in water and titrated to approximately pH 7.2 with a few drops of 1N HCl. Haloperidol, sulpiride and HA-966 were each dissolved in a few drops of glacial acetic acid, diluted with deionized water and titrated to pH 6.3; SCH 23390 was dissolved in a 25% solution of propylene glycol. All drugs were injected in a volume of 0.5 ml. Control animals received the vehicle in the same volume as the drug and with the same number of injections. Assay of tyrosine hydroxylase
The rats were killed by decapitation. The adrenals were quickly removed, cooled on ice, freed from capsular tissue and weighed. Whole adrenals were used for determination of the activity of adrenal tyrosine hydroxylase. This was assayed according to the method of Nagatsu, Levitt and Udenfriend (1964), as modified by Gauthier, Gagner and Sourkes (1979). Statistical analysis
Values in all Tables are expressed as means f SE. Student’s t-test was used for the comparison of paired means (Snedecor, 1956). In most experiments a two-tailed t-test was performed. However, when the direction of effect of a particular treatment was already established and the effect of a further drug on that vector was to be determined, a one-tailed t-test was often applied. For two-way experiments (drug A x drug B) and three-way experiments (drug A x drug B x replications), the analysis of variance (ANOVA) was used (Snedecor, 1956). RESULTS
Effect of apomorphine
Initially it was necessary to determine the relationship of the activity of adrenal tyrosine hydroxylase to the dose of apomorphine. From the results in Figure 1, it is evident that tyrosine hydroxylase was induced over a wide range of doses of apomorphine but that as little as 0.1-0.2mg/kg was effective. In the next experiment, the ability of two postsynaptic dopamine-receptor blockers, namely SCH23390 and I-sulpiride (Iorio, Barnett, Leitz, Houser and Korduba, 1983; Honda, Satoh, Shimonura, Satoh, Noguchi, Uchida and Kato, 1977), to abolish the induction caused by apomorphine was tested. In this case, apomorphine in a dose of 3 mg/kg, was injected 30 min after the administration of SCH23390 (3 mg/kg i.p., twice daily) or I-sulpiride (40 mg/kg S.C.once daily) to separate groups of rats. In the doses used, neither of these antagonists alone
Control
of
523
adrenal tyrosine
received apomorphine, SCH23390 caused decreases in the mean level of activity of adrenal tyrosine hydroxylase, but sulpiride did not do so (Table 1). Efect
of dopamine autoreceptor agonists
In order to test further the possibility of an action at autoreceptors, drugs that are reported to be specific for dopamine autoreceptors were used in the next set of experiments. The first compound tried was 3-(3hydroxyphenyl)-N-n-propylpiperidine [( + )3-PPP], a compound that binds presynaptic DA receptors and inhibits the release of dopamine (Hjorth, Carlsson, Wikstrom, Lindberg, Sanchez, Hacksell, Arvidsson, 0 0.1 0:2 0:4” 018 ” ,:s Svensson and Nilsson, 1981). It was administered to APOMORPHINE (mg/kgl rats in a dose of 10 mg/kg (s.c.), once daily for 3 days. Fig. 1. Effect of apomorphine on the activity of adrenal As shown in Table 2, this treatment elicited a tyrosine hydroxylase. Values are mean + SE of indicated significant inductive effect on the activity of adrenal number of animals in the brackets. Apomorphine was tyrosine hydroxylase. The intracerebroventricular dissolved in saline and the animals received the indicated administration of (+)3-PPP in the amount of amount in a volume of 0.5 ml (s.c.), four times a day for 3 days. Animals were killed on the fourth day. aP
I. Effect of SCH 23390 and sulpiride on apomorphine-elicited increase in the activity of adrenal tyrosine hydroxylase Activity of tyrosine hydroxylase
Treatment Apomorphine Apomorphine + SCH 23390 Apomorphine Apomorphine + sulpiride
55.8 f 41.1 f 58.5 f 50.3 f
4.75 (8) 2.84 (9)’ 4.41 (8) 4.61 (8)
The activity of tyrosine hydroxylase is expressed as nmol dihydroxyphenylalanine (DOPA) formed per hr per pair of adrenals. Values are mean f SE for the indicated number of animals. Drugs were administered as follows: SCH23390: 3 mg/kp (i.p.) twice daily; sulpiride; 40 mgjkg (s.c.) once daily; apomorphine: 3 mg/kg (s.c.) 30 mitt following the injections of antagonists. The animals were treated for 3 days and killed on the fourth day. ‘P < 0.05 compared to apomorphine-treated rats. Table 2. Effect of ( + )3-PPP and GABAergic drugs on the activity of adrenal tvrosine hvdroxvlase Experiment A B C D E F
Treatment Control ( + )3-PPP (S.C.) Control ( + )3-PPP (i.c.v.) Control Muscimol (s.c.) Control GOBA (i.p.) Control HA-966 (i.p.) Control HA-966 (i.c.v.)
Activity of tyrosine hydroxylase 33.4 k 42.9 + 25.7 f 32.7 f 31.6 f 59.7 + 33.1: 41.2+ 24.8: 35.1 + 39.2 I 51.5 f
2.48 (9) 2.34’ (IO) I .98 (7) I .74’ (9) 2.05 (23) 1.97**(25) 1.4(14) 1.31**(16) 1.12(15) 1.08**(16) 3.25 (8) 3.76*** (6)
Enzyme activity is expressed as in Table I. The drugs were injected as follows: ( + )3-PPP: IOmg/kg once daily for 3 days; for the intracerebroventricular i.c.v.) experiment with (+ )3-PPP, one injection of 2 pg/rat was given. y-Hydroxybutyrate (GOBA): 0.5mg/kg once daily for 3 days; HA-966: I SOmg/kg once daily for I day; muscimol: 3 mg/kg twice a day for 3 days; for the inttacerebroventricular (i.c.v) experiment with HA-966, one injection of 5 pg per rat was given. All animals were killed on the fourth day. Values are mean + SE of indicated number of animals. Probabilities for comparison with saline controls are: *P < 0.05; **P < 0.01; ***p < 0.005.
524
R. GABORet al. Table 3. Effect of HA-966 on denervated adrenal tyrosine hvdroxvlase Activity of tyrosine hydroxylase” Treatment Hemi-splanchnicotomy Hemi-splanchnicotomy
+ saline + HA-966
Innervated
Denervated
19.9 + 3.26 (7) 40.9 f 2.88** (9)
21 .Ok 4.32 (4) 16.2 + 3.26’ (7)
-
‘Per single gland. The HA-966 was given in a dose of I50 mg/kg once daily for 3 days by the intraperitoneal route and animals were killed on the fourth day. Two experiments were carried out and the results were subjected to ANOVA. This yielded a mean square for error of 74.5234 (I9 d.f.) which was used for estimating the SE of means of various sizes and of differences between the means. *Not significantly different from the means for control adrenals (P z 0.05). **Significantly different from the other means (P < 0.005).
specific than ( + )3-PPP for presynaptic receptors and is devoid of activity on postsynaptic receptors (Anden, Nilsson, Ros and Thornstrom, 1983). It was injected in doses of 1, 2 and 3 mg/kg (s.c.) once daily for 3 days. The activity of adrenal tyrosine hydroxylase increased in a dose-dependent fashion as a result of this treatment (Fig. 2). Effect of GABAergic drugs
The influence of a GABA system on the induction of the adrenal enzyme was studied by measuring the activity of adrenal tyrosine hydroxylase after the injection of various GABAergic agents. The first drug tested was muscimol, a GABAA receptor agonist. This, when administered to rats in a dose of 3 mg/kg (s.c.) produced a very significant increase in the activity of adrenal tyrosine hydroxylase (Table 2). Sodium y -hydroxybutyrate and HA-966, two analogues of GABA, which temporarily prevent the release of dopamine from nigrostriatal fibres (Walters, Roth and Aghajanian, 1973; Hillen and Noach, 1971), were also tested for their effects on the activity of the adrenal enzyme. As shown in Table 2, y -hydroxybutyrate given once daily for 3 days by the intraperitoneal route caused a significant induction of adrenal tyrosine hydroxylase. In the case of HA-966, a single dose, given intraperitoneally, caused a mean increase of 42% in the activity of adrenal tyrosine
hydroxylase, as measured 96 hr later. Because of the pronounced effect of HA-966, this compound was tested by single intracerebroventricular injection. in the amount of 5 pgg/rat, the animals being killed on the fourth day (96 hr later). Again, as seen in Table 2, a very significant induction of adrenal tyrosine hydroxylase was observed. In order to confirm the central action of the GABAergic drugs, HA-966 was tested in rats that had one adrenal gland denervated some days before the treatment with drugs began. In these animals, the nerve input to the left adrenal gland was eliminated by splanchnicotomy; the innervation of the right gland remained intact. The results from such an experiment are shown in Table 3. The HA-966 caused a large increase in the activity of tyrosine hydroxylase of the innervated gland, but was ineffective on the denervated side. Interaction between GABAergic drugs
The ability of dopamine postsynaptic-receptor blockers to abolish the induction of adrenal tyrosine hydroxylase by GABA analogues was tested by the use of three dopamine antagonists, namely haloperidol, I-sulpiride and SCH23390. These drugs were injected 30min prior to the administration of the GABAmimetics. None blocked the induction of adre-
Table 4. Effect of DA blocking agents on the induction of adrenal tyrosine hydroxylase, caused bv GABAernic druns Exoeriment A
B
C
Treatment
Activity of tyrosine hydroxylase
Sulpiride GOBA GOBA + Sulpiride HA-966 HA-966 + Sulpiride
46.1 *3.16(S) 45.3 f 3.16(S)* 34.2 f 1.86 (7) 32.4 f 1.74 (8)’
SCH 23390 HA-966 HA-966 + SCH 23390
26.8 k 2.4 (7) 28.5 f 2.24 (8)’
Haloperidol Muscimol Muscimol +
and dopaminergic
53.5 i 6.92 (5) 68.4 i 5.49 (6)’
Halooeridol Drugs were administered as follows: GOBA: 0.5 g/kg (i.p.) once daily for 3 days; HA-966: I50 mg/kg (i.p.) for I day; muscimol: 3 mg/kg (s.c.) twice daily for 3 days: sulpiride: 40 mg/kg (s.c.) once daily 30 min prior to GABA drugs; SCH 23390: 3 mg/kg, (i.p.) twice daily, 30 min prior to HA-966; haloperidol; 5 mg/kg, (i.p.) once daily, 60 min prior to muscimol. Animals were killed on the morning after the last injection. Values are mean f SE of indicated number of animals. *P > 0.05 for comparison with corresponding control mean (no blocking agent).
Control of adrenal tyrosine Table 5. Effect of HA-966 and BHT 920 on the activity tyrosine hydroxylase Treatment Control HA-966 BHT 920 HA-966 + BHT 920
Activity
of tyrosine
25.1 37.2 36.0 49.6
of adrenal
hydroxylase
f 2.59 (3) (100%) + 1.47” (4) (149) : I .65* (4) (143) _+ 3.85” (5) (198)
The drugs were injected as follows: HA-966: 100 mg/kg, (Lp.) once daily for 3 days; BHT 920: 1 mg/kg, (s.c.) once daily for 3 days; for animals receiving both drugs, the same amount of drug was given 30 min apart. All animals were sacrificed on the fourth day. Values are mean f SE of indicated number of rats. Probabilities for comparison with saline controls are: *P < 0.025; l*P < 0.005.
nal tyrosine hydroxylase produced by the GABAergic substances (Table 4). If GABAergic drugs and dopamine autoreceptor agonists both act by inhibiting the release of dopamine, then the administration of these drugs together, at submaximal doses, should produce an additive response. This was tested by giving rats HA-966 (100 mg/kg) and BHT 920 (1 mg/kg), 30 min apart, daily for 3 days. The results in Table 5 show that the mean increase of 98%, obtained when the drugs were given together, was precisely an additive effect. DISCUSSION
Earlier work from this laboratory established the involvement of central dopaminergic systems in the induction of adrenal tyrosine hydroxylase (Quik and Sourkes, 1976). The large doses of apomorphine (3-lOmg/kg), used in earlier work, caused stimulation of both pre- and postsynaptic receptors and thus were not discriminatory. From the present work (Fig. l), it was evident that doses as small as 0.14.2mg/kg of apomorphine were effective in inducing adrenal tyrosine hydroxylase and that larger doses produced no further effect. In the small doseinteracts mainly with prerange, apomorphine synaptic dopamine receptors and thereby inhibits the release of dopamine (StPhle and Ungerstedt, 1984; Zetterstriim and Ungerstedt, 1984). The involvement of presynaptic receptors was further substantiated by the use of postsynaptic dopamine-receptor blockers. The two drugs used, namely SCH23390 and lsulpiride, produced different effects. The failure of sulpiride LO affect significantly the action of apomorphine, points to the role of a presynaptic component in the induction of the enzyme. The inhibitory action of SCH23390 in this test seemed to illustrate the fact that this compound can interact with both D, and D, receptor types, including Dz autoreceptors of the type found predominantly at dopaminergic nerve endings (Creese, 1987; Goldstein, Litwin, Sutton and Malick, 1987; Morelli and Di Chiara, 1985; Plantje, Daus, Hansen and Stoof, 1984). Because the use of postsynaptic receptor blockers did not provide conclusive evidence for the involvement of presynaptic receptors, two drugs that
525
are reported to be specific for autoreceptors were used. Both drugs, namely ( + )3-PPP and BHT-920, proved to be effective in inducing adrenal tyrosine hydroxylase (Table 2; Fig. 2). The effect of BHT-920 was dose-dependent, as analyzed by multiple regression analysis (Fig. 2). This substance is said to be more specific than (+)3-PPP for autoreceptors and is devoid of activity at postsynaptic receptors (Anden, Nilsson, Ros and Thornstrom, 1983). These experiments, then, demonstrate clearly that the inhibition of the release of dopamine by stimulating autoreceptors results in the induction of adrenal tyrosine hydroxylase. The site of action of these drugs could be at the level of the neural loop formed by the strionigral and nigrostriatal tracts. A GABAergic system is known to provide feedback from the striatum to the substantia nigra and the activation of this system prevents nigrostriatal neurons from releasing dopamine (Walters et al., 1973; Hillen and Noach, 1971). Various GABA agonists, at least one of them (HA-966) now shown to act centrally, produced significant induction of adrenal enzyme in a manner similar to that of the dopamine autoreceptor agonists (Table 2). Because dopamine postsynaptic receptorblockers did not abolish the induction produced by GABA drugs (Table 4), postsynaptic stimulation is probably not involved in this type of induction. As mentioned earlier, if GABAmimetic drugs modulate the dopaminergic system, then drugs acting at the autoreceptors should be able to affect the GABAmediated induction. As there is no dopamine autoreceptor-antagonist available that can block specifically the presynaptic receptors, it was decided to follow a different approach: if GABAergic drugs and dopamine autoreceptor agonists both act by inhibiting the release of dopamine, then the adminis-
,
I
0
2
1 BHT
920
I
3
(mg/kgl
Fig. 2. Effect of BHT 920 on the activity of adrenal tyrosine hydroxylase. Values are mean f SE of 5 animals. The BHT 920 was dissolved in saline and the indicated amount was injected once daily in a volume of 0.5 ml (s.c.) for 3 days. Animals were killed on the fourth day. The slope (b + SE = 11.69 f 2.4) is significantly different from zero (P < 0.001, 18 degrees of freedom), as analyzed by multiple regression.
R. GABOR etal.
526
tration of such drugs together at submaximal doses should produce an additive response. The results from such an experiment (Table 5) show precisely an
additive effect when HA-966 and BHT 920 were given together. These observations provide conclusive evidence that stimulation of either a central GABA system or a dopamine autoreceptor can produce similar responses. Hitherto, it has been established that the central dopaminergic and cholinergic systems play an excitatory role, whereas (5hydroxytryptamine, 5-HT) contributes inhibitorily to the induction of adrenal tyrosine hydroxylase (Sourkes, 1985; Lewander et al., 1977). The present work has revealed a central GABAergic pathway which exerts a net excitatory influence on the induction of this enzyme. The similarity of inductive effects of small amounts of apomorphine dopamine-autoreceptor agonists and analogues of GABA, combined with the fact that GABA inhibits the nigrostriatal DA system, now suggests that the DA-elicited increase in adrenal tyrosine hydroxylase is a presynaptically mediated phenomenon. The fact that a GABA agonist and a DA autoreceptor agonist had an additive effect (Table 5) points to the striatonigral GABAergic feedback pathway as of importance in the control of the nigrostriatal dopaminergic system. The role of non-dopaminergic pathways in the regulation of the activity of adrenal tyrosine hydroxylase is demonstrated by the failure of dopamine antagonists to block the induction caused by analogues of GABA (Table 4). One such site of action of GABA has been discussed above. However, the presence of GABAergic neurons in the raphe nuclei, where they exert inhibitory control over the serotonergic cells therein (Nishikawa and Scatton, 1985; Belin, Nanopoulos, Didier, Aghera, Steinbusch, Verkofstad, Maitre and Peyot, 1983), opens up another possible site of action of GABA drugs in inducing the adrenal enzyme. Acknowledgements-This
work has been supported by a
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