Naunyn-Sehmiedeberg's Arch. Pharmacol. 291, 193--200 (1975) �9 by Springer-Verlag 1975

The Regulation of Striatal Tyrosine Hydroxylase Effects of Gamma Hydroxybu t r i c Acid and Haloperidol

B. Zivkovic*, Alessandro Guidotti, and Erminio Costa

Laboratory of Preclinical Pharmacology, National Institute for Mental Health, Saint Elizabeths Hospital,

Washington, D. C.

Received June 26 / Accepted August 28, 1975

Summary. Gamma-hydroxybutyric acid (GHBA) in doses that increased the striatal dopamine (DA) content of rat brain failed to increase the affinity of striatal tyrosine hydroxylase (TH) for its pterdine eofactor or to change the sensitivity of the enzyme to the inhibition by DA. Haloperidol (1 mg/kg) decreased the apparent Km of striatal TH for the pteridine eofactor. However, when GHBA was injected before haloperidol it prevented the decrease in the apparent Km of TH, in a dose related manner. In vitro GHBA (10 -~ M) neither changed the stimulation of the striatal adenylyI eyclase by DA nor its inhibition by haloperidol.

These results suggest that in striatal dopaminergic terminals the Km of TH for the pteridine cofactor is regulated by an molecular mechanism which requires that the impulse flow in the DA neurons is unimpaired.

Key words: Tyrosine hydroxylase -- Km for p~eridines -- Haloperidol --~-HIy- droxybutyric acid.

The injection of antipsychotic drugs in rats promptly decreases the apparent Km of striatal tyrosine hydroxylase (TH) for two synthetic pteridine cofactors (Zivkovic et al., 1974), in striatum (Costa et al., 1974) and nucleus accumbens (Zivkovic et al., i975a) this change is dose re- lated. Similar findings have been reproduced by Murrin et al. (1974) and Lovenberg and Bruckwiek (1975) using the naturally occurring cofactor, tetrahydrobiopterin.

Apomorphine and piribedil (ET 495) fail to change the kinetic state of TH, but they readily antagonize the decrease of the Kra for the pteri- dine cofactor elicited by antipsychotics (Zivkovic et al., 1974; Zivkovic and Guidotti, 1974). Such an interaction between agonists and blockers of DA postsynaptic receptors, suggests tha t postsynaptie DA receptors may participate in the regulation of the kinetic state of striatal T H (Zivkovic et al., 1975b). Since the antipsyehotics increase and the DA receptor agonists decrease the firing of DA neurons in the zona compacta

Send o]]print requests to: E. Costa, Laboratory of Preclinical Pharmacology, National Institute of Mental Health, W. A. White Building, St. Elisabeths Hospital, Washington, D. C. 20032, U.S.A.

* Present address: Dept. of Biology and Experimental Medicine, Institute Ruder Boskovie, YU-41001 Zagreb, Yugoslavia.

13"

194 B. Zivkovic et al.

of substantia nigra (Bunney et al., 1973a, b), it was inferred that the kinetic state of TH in DA terminals is regulated by the frequency of nerve impulses (Zivkovic et al., 1974). Moreover, the possibility that autoreceptors in DA neurons regulate the function of DA nerve terminals (Carlsson, 1975) has invited speculation on the role of these autoreceptors in the control of striatal TH. The present report fails to support this speculation by demonstrating that haloperidol does not change the ki- netic state of striatal TH when nerve impulse flow is decreased by gamma hydroxybutyric acid. Since haloperidol also fails to activate striatal TH after hemisection (Zivkovic et al., 1975c) these data cannot easily be reconciled with the hypothesis that autoreceptors in DA terminals are the only regulatory mechanisms for striatal TH.

Materials and Methods Male Sprague Dawley rats, 150 g (Zivic Miller, Allison Park, Pa.) were housed

for at least 5 days in our animal quarters. The rats were kept at 24~ with alter- nating periods of light (14 hrs) and darkness (10 hrs). The animals were allowed free access to water and standard laboratory chow until 2 hrs prior to the experiment.

Gamma hydroxybutyric acid (GHBA) (Aldrich Chemical Co., Milwaukee, Wis.) was dissolved in distilled water. Haloperidol base (McNeil, Fort Washington, Pa.) was dissolved in a few drops of glacial acetic acid and diluted to the desired volume with saline after adjusting the pH to 5--6 with 1 N NaOH.

Rats were killed by decapitation at different time intervals after saline and/or drug treatment as indicated in the Results. Striata were rapidly dissected and frozen on dry ice. The TH activity was assayed in striata homogenized in 0.05 mM tris acetate buffer (pH6.0) containing 0.2~ Triton • These homogenates were centrifuged at 15 000 • g for 10 min and TH activity measured in an aliquot of the supernatant by a modification (Zivkovic et al., 1974) of the method of Waymire et al. (1970). The activity of adenylyl cyclase in striatal homogenates and its stim- ulation by DA was measured as reported by Kebabian et al. (1972). The concen- tration of striatal DA was measured as described by Neff et al. (1971) that of protein by the method of Lowry eta'. (1951). The statistical significance of the differences in unbiased mean values was calculated by the 2-tailed Student's "t" test.

Results GHBA (10 -4 M) added to homogenates of striatum changed neither

the adenyl cyclase activity nor the stimulation of this enzyme by DA (5 • 10 -6 M). ttaloperidol (10 -8 M) inhibited the stimulation of adenylyl cyelase by DA (5 • 10 -8 M) in the presence and absence of GHBA (10-4M) Moreover, the affinity of TH for DMPH 4 (Kin 0.87 raM) was similar in the presence or absence of GHBA (10 -4 M). This concentration of GHBA also failed to change the affinity of striatal TH for pteridine when added to striatal homogenates from rats previously treated with haloperidol (lmg/kg i.p.).

GHBA (750 mg/kg i.p.) produced a significant increase of striatal DA content within 15 rain; at 1 hr after GHBA injection the DA content was almost doubled (Fig. 1). When striatal TH activity was measured in

The Regulation of Striatal Tyrosine Hydroxylase 195

12 ~ I * II -t

II ~ / I0 ~ 9o3r~

a 9 8 ~ o z~ .d0pamlne ~

~ 3 ~" ~ ' )

"" 7 6N m o ~ 5...-i 6 5 ~

b ,b 6J minutes ofter GHBA

Fig. 1. Dopamine content and tyrosine hydroxylase activity in strJata of rats at different time intervals after the intraperitoneal injection of gamma-hydroxy- butyric acid (GHBA) (750 mg/kg). Tyrosine hydroxylase was measured in the presence of 0.4 mM DMPH 4. Each point is the mean =~ S.E.M. (vertical bars) of

5 ra~s, * P < 0.05 when compared with rats receiving saline (0 time)

the presence of nonsaturating concentrations of DMPItt (0.4 raM) there was no change in the enzyme activity during the first hr after the in- jection of GHBA (750 mg/kg i.p.) (Fig. 1). The apparent Km of striatal T H for DMPI-It in saline and GHBA (750 mg/kg i.p.) t reated rats was 0.87 mM and 1.1 mM, respectively.

The apparent Km with respect to L-tyrosine was 0.090 mM in saline and 0.080 mM in GHBA treated rats. In rats pretreated with saline, haloperidol (1 mg/kg i.p.) produced about a 4-fold decrease in the ap- parent K~ of striatal TH for DMPHa, whereas in rats receiving GHBA (750 mg/kg i.p.) 30 min before haloperidol (1 mg/kg i.p.) the K~n of TH for DMPIt 4 was comparable to that in rats receiving only saline (Fig. 2).

A pretreatment of rats with GHBA antagonized the increase of TH activity elicited by haloperidol in a dose-related manner (Fig. 3). This antagonism was estimated by measuring TH activity in the presence of DMPI~ 4 concentrations below saturation (0.4 raM). Haloperidol (1 mg/kg i.p.) produced a smaller increase in striatal TH activity in rats pre- t reated with GHBA (350 mg/kg i.p.) than in rats pretreated with saline ; moreover, in rats receiving 750 mg/kg of GHBA, 1 mg/kg i.p. of halo- peridol failed to increase striatal TH activity (Fig. 3).

The effects of different concentrations of dopamine on the initial velocity of dope formation catalyzed by striatal homogenates incubated with various concentrations of DMPH 4 are reported in Fig.4. The ad- dition of various concentrations of dopamine to TI-I preparation of striata from saline or GItBA treated rats suggested that this amine competitively interferes with the I)MPH v In the presence of various dopamine con-

196 B. Zivkovie et al.

0,6

0.4 ! V

0,2

Krn (raM) ~ / �9 HALOPERIDOL 0.20 / ~

.,0o o.,. / /

-4 -2 o 2 4 ~ 8 io ,~ ,~ I / DMPH4 ml~ I

Fig.2. Double reciprocal plots of the initial velocities of tyrosine hydroxylation versus various concentrations of DMPH 4. The concentration of tyrosine was 0.2 mM. Rats received an intraperitoneal injection of gammahydroxybutyric acid (GHBA) 30 rain before haloperidol (1 mg/kg i.p.). Animals were killed 30 min after halo-

peridol. Velocity is expressed as nmoles of CO s formed per hour per mg protein

14

F- -~ 12 r- {J ~ . I0 ~ 2

I I : . c

>- e,I

bJ ~ Z w

o E

~ 2

0 SALINE GHBA GHBA GHBA

I00 350 750

Fig. 3. Effect of haloperidol on tyrosine hydroxylase activity in rats pretreated with various doses of gammahydroxybutyric acid (GHBA). Doses are in mg]kg i.p. Rats received GHBA in~raperitoneally 30 rain before haloperidol (1 mg/kg i.p.), they were killed 30 rain after haloperidol injection and tyrosine hydroxylase was assayed in presence of 0.4 mM DMPH~. Each bar is the mean of 5 animals: brackets represent S.E.NL * _P < 0.05 as compared with rats receiving haloperidol after saline or only saline. ** P < 0.01 as compared with rats receiving only

GHBA or saline

The Regulation of Striatal Tyrosine IIydroxylase 197

DOPAMINE ( r a M ) SALINE

0 o.o �9 o.o5 �9 o.I

K m = 0,87 mM /

DOPAMINE (raM) GHBA

0 o.0 �9 0.05

�9 O,I 3

I V 2

I

Km~O.84mM

_;, , ~ I/DMP% mM

Fig. 4. Effect of two concentrations of ])A added to striatal tyrosine hydroxylase preparations from saline and GHBA treated rats on the double reciprocal plot of initial velocities versus various concentrations of DMPH 4. GttBA (750 mg/kg i.p.) was injected 1 hr prior k) death. The concentration of tyrosine in the assay was

0.1 mM. Enzyme velocity is expressed as nmoles of CO~ hr/mg protein

centrations the double reciprocal plots of the enzyme velocities vs. DMPH concentration yielded parabolic lines. Therefore, the Kt for dopamine cannot be readily estimated. However, the data of Fig.4 show that the inhibition produced by a given DA concentration is the same in saline and GHBA treated rats.

D i s c u s s i o n

Several lines of evidence demonstrate that Gt tBA blocks nerve impulse flow in nigro striatal dopaminergie neurons (Gessa et al., 1968; Roth et al., 1973; Stock et al., 1973) and increases the DA content of

198 B. Zivkovie et al.

striatum (Gessa et al., 1968). More recently, it has been reported that in dopaminergie neurons the decrease of neuronal activity is associated with a marked increase in the affinity of striatal TH for tyrosine or DMPHa (Roth et al., /975). The present experiments confirm that the injection of GHBA increased the striatal DA content by more than 60 ~ in 1 hr. However, the TH affinity for the pteridine cofactor or for tyrosine fails to increase under our experimental conditions. At the present time, we cannot readily reconcile the discrepancy between our findings and the report by Roth and colleagues (1975). The conditions for measuring the apparent Km of TIt were different in our experiments than in those of Roth and colleagues (1975). We measured the Km in the presence of saturating concentrations of either substrates or cofactors. In the experi- ments by Roth et al. (1975) saturating conditions were not always fol- lowed. Probably for analogous reasons also the report by Roth et al. (1975) that the affinity of striatal T H for tyrosine is increased when the impulse flow in striatal neurons is accelerated (i.e., after haloperidol injection) could not be confirmed (Kuczenski and Mandel, 1972; Ziv- kovie et al., 1974; Lovenberg and Bruckwiek, 1975).

Doses of haloperidol which usually decrease the Km of TH for DMPH~ fail to change the TH affinity for pteridines when given after GHBA.' Since haloperidol increases DA synthesis because it enhances the affinity of striatal TH for the pteridine eofactor (Zivkovic et al., 1975a; Carenzi et al., 1975) the results of the present experiments provide an explana- tion for other reports showing that GHBA blocks the increase of striatal homovanillic acid elicited by haloperidol and chlorpromazine (Roth, 1973). Since various drugs may regulate striatal TH (Roth et al., 1975) by acting on striatal DA autoreccptors (Carlsson, 1975), we studied whether GHBA which antagonized the decrease in the Km of striatal T H elicited by haloperidol acted on DA autoreceptors. We have shown that GHBA changed neither the stimulation of adenylate cyclasc by DA nor its inhibition by haloperidol. Moreover, GHBA did not decrease the affinity of T H for pteridines when added to striatal homogenates of rats receiving saline or haloperidol. Since GHBA reduces the flow of nerve impulses through nigro striatal dopaminergic neurons when injected into the substantia nigra but not when injected into the striatum (Anden and Stock, 1973), one might infer that the GHBA receptors are located in the pars compaeta of substantia nigra. Although GHBA causes an accumu- lation of DA it does not change the kinetic properties of TH nor, in our hands, changed the TIt inhibition by DA. Thus, if GHBA decreases the release of DA extraneuronally, this decrease does not affect the kinetic properties of TH in nerve terminals. This finding implies than when little or no DA reaches presynaptic autoreeeptors the affinity of TH for the pteridines remains unchanged. Such a conclusion is supported by results obtained in rats with cerebral hemisection and injected with haloperidol

The Regulation of Striatal Tyrosine Hydroxylase 199

(Zivkovic et al., 1975e). We therefore suggest that haloperidol decreases the Km of striatal TH for pteridines, only when the nitro striatal path- way is intact and the antipsyehotic can increase the impulse flow in DA neurons. Thus, in our view haloperidol changes striatal TH independently from its action on DA autoreceptors (Carlsson, 1975).

I t is also our opinion that if the molecular mechanisms that regulate the Km of striatal TH include an activation of cyclic AMP dependent protein kinase which phosphorylates TH or another regulatory protein (Morgenroth et al., 1975) this phosphorylation is not regulated by DA autoreeeptors. Since antipsychotic drugs lower the affinity of TH for pteri- dines but block DA activation of adenyl cyclase it appears improbable that by blocking DA autoreceptors, they activate the cAMP dependent phosphorylation of TH that regulates TH activity (Morgenroth et al., 1975). I f a cAMP dependent phosphorylation is operative in the modul- lation of striatal TH, the activation of the protein kinase involved may result from an increase in impulse flow rather than from the activation of hypothetical DA antoreceptors (Carlsson, 1975) at dopaminergie termi- nals.

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