European Jonrnal of Pharmacologo" - Molecular Pharmacology Section.

188 (1990l 123-128

123

Elst.vier EJPMOL 90061

Effects of pertussis toxin on inhibition of synaptosomai tyrosine hydroxylase activity by apomorphine K. H a r a d a t g. Meller z and M. Goldstein 1 New York Universio" Medical Center. Neurodwmistrv Research L~borat~;rie~ I and .~liilhau.~er Lahtwdtormx :. New ~~r~. N Y 10016, U.S.A.

Received 7 November1989. accepted 14 No~ember1989

The effects of pertussis toxin (PTX) on synaptosomal tyrosine hydroxylase {'I-H) activity and on ~he inhibition of synaptosomal TH activity by apomorphine were investigated. Exposure of striatal synaptosomes to PTX does not affect basal- or forskolin-stimulated TH activity, bat attenuates apomorphine-elicited inhibition of forskofin-stimulated synaptosomal TH activity. There is a good correlation between the attenuation oi apomorphine-elicited inhibition of synaptosomal TH activity by PTX and (-)-sulpiride. suggesting that G proteins are involved in ~he dopamine (DA) autoreceptor-mediated regulation of the enzyme activity. The exposure of synaptosome to PTX results in a 40-50% inactivation of Gi and Go proteins, which is evident from ,he reduction of ADP ribosylation with [3zP]NAD of the remaining G proteins following preincubation with the to: in. The present study also demenstrates that striatal synaptosomal preparations can be used for investigations of the molecular properties of nerve terminal DA autoreceptors. Dopamine au'.oreceptor: G proteins: Tyrosine hydroxylase (inhibition)

1. Introduction Dopamine (DA) receptors involved in the regulation of DA synthesis (DA autoreceptors) have been extensively studied in synaptosomal preparations (Christiansen and Squires. 1974; Bronaugh and Goldstein, 1975). Stimulation of these receptors by DA agonists leads to the inhibition of synaptosomal tyrosine hydroxylase (TH) activity, while DA antagouists reverse this effect. The validity of inhibition of synaptosomal TH by some DA a3onists as an index of stimulation of DA autoreceptors has been questioned since the inhibition was not reversed by DA antagonists

Correspondence to: Menek Goldstein. Ph.D.. Neurochemistry Research Laboratories. New York University McdicM (.'enter. 560 First Avenue. Rm. H-544. New York. NY 10016. U.S.A.

(Fox~Ier et al.. I985). However. it recently was reported that activation of synaptosomat TH by forskotin was potently inhibited by DA agonists. at,d that this effect was reversed by DA antagonists (Strait and Kuczenski. 1986). We have recently reported similar results in striatal slices (Bohmaker et al.. 1989). The~e findings imply that the forskolin-indu:ed increase in phospborytation and activation c~f synaptosomat TH is regulated by DA autorecepto~ s. Pharmacologicai studies suggest that DA autoreceptors resemb',e the DA D-2 receptor subtype (Goldstein. 1984). DA D-2 receptors are coupied negatively to adenylate cyclase via the guanine nucteotide regulatory proteins G, a ~ d / o r G,,. Pertussis toxin inactivates G, and G,, proteins by ADP ribos?hdon of their a subunits and thereby inhibits receptor-mediated respoases in systems in which the receptor is linked to the effector (Weiss

0922-4106/90/$03.50 ,~' 1990 ElsevierScience Publishers B.V.(Biomedica| Divisioal

124 et al., 1985). !n order to further characterize the synaptosomal striatal DA autoreceptors which regulate DA synthesis we have investigated the inhibitory action of apomorpbine on the forskolin-elicited activation of synaptosomal TH activity, In addition, we have examined whether pertussis toxin (PTX), which uncouples GTP binding proteins (G i and Go) from some neurotransmitter receptor, alters the apomorphine-elicited inhibition of synaptosomal TH activity. The effect of synaptosomal exposure to PTX on the ADF ribosylafon of G proteins was measured by ADP ribosylation with [32p]NAD of the remaining G proteins following preincubation with the toxin.

2, Materials and methods

( - ) - A p o m o r p h i n e hydrochloride and ( - ) sulpiride (Research Biochemicals, Inc., Natick, MA), PTX (List Biological Labs, Campbell, CA). dithiothreitol, forskolin, m-hydroxybenzylhydrazine (NSD-1015) and soybean trypsin inhibitor (Sigma, St. Louis, MO), sodium dodecyl sulphate (SDS) (Pierce, Rockford, IL) [32p]nicotinamide adenine dinucleotide ([32p]NAD) (New England Nuclear, Boston, MA), adenosine triphosphate (ATP) and guanosine triphosphate (GTP) (Boehringer Mannheim Biochemicals, Indianapolis, IN) were obtained from the indicated suppliers. All other chemicals used were standard laboratory reagents of analytical grade. Forskolin and ( - ) sulpiride were initially dissolved in t:thanol and 10% acetic acid. respectively, and then diluted with 20 mM HEPES buffer. Male Sprague-Dawley rats (150-200 g, Taconic Farms, Germantown, NY) were decapitated and the brains rapidly removed and placed on ice. The striata were immediately dissected and homogenized in 20 volumes (v/w) of 0.32 M sucrose at 4 ° C (except for the experiment with pertussis toxin where five volumes (v/w) were used), Synaptosomal TH activity was determined by measuring striatal L-DOPA accumulation following inhibition of L-aromatic amino acid decarboxylase (AAD) with NSD-1015. The following incubation procedure was carried out. First, 100 #1 of homogenate (5 mg of original tissue) was

mixed with 510 pl 29 mM HEPES buffer containing 10 -7 M forskolin and (mM): NaCI i50; KCI 5.0:MgSO4 1.0; NaHzPO 41.2; CaCI 2 1.0; ascorbic acid 0.2; glucose 10.0; saturated with ~5% 0_,/5% CO~; pH 7.2 ill glass test tubes. Incubation at 37°C was started following addition of 70 #1 of various concentrations of apomorphine or vehicle, and 5 min later 20 /.tl of NSD-1015 was added (final concentration 5 x 10 -5 M). Incubation was carried out for an additional 15 min and stopped by adding 300 pl of ice-cold 0.3 M perchloric acid (PCA) containing 8 ng of a-methyI-DOPA as internal standard. To determine the effects of PTX the following preincubation procedures were carried out. 300 #1 of homogenate was mixed with 100-200 #1 PTX or velficle and 100 ttl of dithiothreitol (final concentration 1.0 mM). The total volume was adjusted to 1.2 ml with 20 mM HEPES buffer. The tubes were preincubated for 60 min at 3 0 ° C under a constant stream of 95% 02/5% CO 2. The mixture was then centrifuged at ~'30 × g for 25 min at 4 ° C and the supernatant was discarded. The pellet was restlspended in 1.2 ml of 20 mM HEPES buffer (5 mg of original tissue/100 p l), and 100/.tl of the homogenr, te was mixed with 510 ttl of 20 m:~.~ HEPES buffer containing 10 -7 M forskolin. Subsequently the incubation was carried out as described above. At the end of the incubation period PCA was added and the tubes were kept in ice-water and sonicated for 15 s. The samples were centrifuged at 16000 × g for 10 min at 4 ° C , and aliquots of supernatant were used for DOPA assay by high pressure liquid chromatography (Michaud et al., 1981). ADP ribosylation was measured as an indication: of inactivation of G proteins (Bean et al., 1988; Nestler et al., 1989). Synaptosomes preincubated with PTX (16 p g / m l ) or vehicle were centrifuged and resuspended in sonieation buffer containing (raM): Tris 50; MgCI 2 6.0; EDTA 1.0; benzamidipe 3.0; dithiothreitol 1.0; sucrose 5% (w/v); soybean trypsin inhibitor 1 t~g/ml; final pH 8.0. Following centrifugation, the pellets were resuspended in ADF-fibosylation assay buffer containing ~mM): Tri; 100; MgC12 5.0; EDTA 0.8; benzamidine 2.4; thymidine 10; dithiothreitol

125 2.8; isoniazid 10: A T P 2.5; G T P 2.0; sucrose 4% ( w / v ) ; Triton X-100 0.5% ( v / v ) : soybean trypsin inhibitor 0.8 t ~ g / m l ; p H 8.0. The A D P ribosylation was carried out in a m e d i u m c o n t a i n i n g 80 ,at of s y n a p t o s o m a l extracts, 3.5 p.1 (1 ptg) of' PTX a n d 16.5/11 of [32P]NAD (approx. 30 C i / m m o l ) . The mixture was i n c u b a t e d for 60 w i n at r o o m temperature, a n d then 750 /xl of i.e-cotd sonication buffer was added. The m i x t u r e was centrifuged (10000 x g for 15 rain) a n d the pellet was resuspended in 30 /tl of 40 m M Tris-HCl buffer ( p H 6.8) c o n t a i n i n g 1 m M dithiothreitol and 2% ( w / v ) SDS. U p o n incubation for 5 rain at 7 0 ° C . 20 p.I of 100 m M N - e t h y l m a l e i m a i d e was added. F o l l o w i n g an a d d i t i o n a l 15 m i n incubation at r o o m temperature, 50 #I of 40 m M Tris-HCl buffer (pH 6.8) c o n t a i n i n g 50% ( w / v ) SDS a n d 6% ( v / v ) fl-mercaptoethanol was a d d e d and the samples were boiled for 2 min. The samples were then subjected to o n e - d i m e n s i o n a l SDS-polya c r y l a m i d e gel electrophoresis with 9% ( w / v ) a c r y l a m i d e / 0 . 2 5 % ( w / v ) bis-acrylamide in the resolving gel (Laemmli. 1970). The resulting gel was dried and exposed to X-ray films for 7-24 h. The b a n d s o,n films were evaluated using a s c a n n i n g densitom~..er ,GS-300. Hoefer Scientific Instruments) in t r a n s m i t t a n c e m o d e a n d expressed as arbitrary u n i t s / r a g protein of s y n a p t o s o m a l ex-

tract. Protein assay was done using dye reagent (Bio-Rad). Statistical significance was calculated using unpaired Student's t-test.

3. Results

3.1. The effect,~ of apomorph#re and ( - )-sulpiride on forskolin-stimulated T H activi(v in seriamt Zvnaptosome In agreement with Frevious reports (Slrait and Kuczenski. 1986: B o h m a k e r et at.~ 1989). forskolin stir.zulated s y n a p t o s o m a ! T H activity a n d ap¢~m o r p h i n e i n h i b i t e d both i3t~al- as well as forskolin-stimulated T H activity to a p p r o x i m a t e l y the same extent. ( - )-Sutpiride had no effect on basalor forskolin-stimuiated TH. and it a t t e n u a t e d the a p o m o r p h i n e - i n d u c e d inhibition of T H activity. However, since ( - ) - s u l p i r i d e more effectively attenuated the f o r s k d i n - s t i m u ! a t e d than basal T H activity (data not shown), we have carried out this s t u d y with the forskolin-stimnlated enzyme. The d a t a presented in table I show that a p o m o r p h i n e inhibited the forskolin-stimulated s y n a p t o s o m a l T H activity in a d o s e - d e p e n d e n t manner. ( - ) Suipiride c o m p l e t e l y blocked the i n h i b i : i c a of T H activity by a tow c o n c e n t r a t i o n of a p o m o r p h i n e .

TABLE l The an,agonism by (-)-sulpiride of apomorphine-induced inh;hifion of TH activ~y in PTX ~16 ~g/mb-treaied and umrea~ed synaptosomes. Data are expressed a~ me~,ns± S.E. (ng/5 mg original tissue) of ,rout expe~qmem~.Preincabanol~ r~'~ed m a partia~ loss of forskolin-stimulated TH activity, but the extent of Ibis lo:,s , , ~ not dependcm on the coaccnrratkm eft PTX {s=e !egcnd, fig 1). ~- )-Sulpiride (M}

DOPA accumula'~mn(~: inhi,.hi~'ion,~syapomo~hme} U mreat~d

PTX -trealed

0

0 {0 ~

5.360 ± 0,0;g9 5,357±004{

3.2(-~9± 0,029 3.2~.8 ~ 0.(~38

5x10 ~

0 10 ~

4%21 +0.082 ~!3.8~ "~ 5A22 ± 0.059 { - 1.6} t,

3A58z0.0H ~{ 5i 3.19! ~0.016 {£~5~

0 10 a

3.874 ± 0.063 {27.7j ~ 5,029_+0.053 {6A I ~~"

2.933 ± 0,0~.8 ;~.6t ~ 296{~= 0.022 t7.g} ~

0 ~0-~

2.6S2 ± 0.044 (50.0'~ ~ 3 g60 ±0.054 (27.9} ~~

1555 ±0.023 ~39.1,~~ 2 Oh9:: 0.025 {35I ~*

Apomorphine (M)

10- v 2x 10 -~'

P < 0.005 compared to corresponding contro| value: ~ P < O.CcJ5compare,~l to ! - ~--sul~;ir~de-ffce?zoup~ al t{~,esame apc~omb~e concentrations.

126 (4) .~50

but only partially attenuated the inhibition of TH activity elicited by higher concentrations of apomorphine,

E ~ Vehicle

i ii PTxm

og/m~~4~/, (4)

3.2. The effects of pertussis toxin on apomorphineeficited inhibition of forskolin-stimulated synaptosomal TH activity

{4~'

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[

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2 x 10-8 10-7 5 x I0 -7 Coneentrotion of (--)opomorphine (M) Fig. 1. Attenuation of apomorphine-induced inhibition on forskolin-stimulated synaptosomal TH activity by pertussis toxin. Striatal synaptosomes were treated with PTX or vehicle for 60 rain at 30°C, centrifuged, and incubated with 10 -7 M forskolin and various concentrations of apomorphine. Control values of DOPA accumulation using synaptosomes treated with vehicle. PTX 8 p.g/ml, or 16 F g / m l (ng/5 mg original tissue) were 2,360_+0.088 (n = 8), 2.288_+0.070 (n = 4) and 2.412_+0.043 (n = 4), respectively. Each value represents the mean from various experiments (n). Significantly different from vehicl~ control: * P < 0.05, * * P < 0.&'31,

A

.o

PTX alone had no effect on TH activity, but attenuated apomorphine-elicited inhibition of forskolin-stimulated synaptosomal TH activity (fig. 1). The effect of PTX was concentration-dependent, and the attenuation was greater when the synaptosomes were exposed to 16 #g/ml instead of 8 /tg/ml of the toxin. The inhibition of synaptosomal TH activity elicited by a low concentration of apomorphine was completely blocked by PTX, while that elicited by higher concentrations was only partially attenuated (fig. 1). Exposure of synaptosomes to PTX abolished the attenuation by (-)-sulpiride of the apomorphinc-induced inhibition of TH activity (table 1).

,°°l

o~

41kD

O

39kD

41kD

vehicle

vehicle

39kD

PTX

P-rx

Fig. 2. The effect of PTX pretreatment on ADP ribosylation of G proteins in striatal synaptosome. Synaptosomes were exposed to F I X