Influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase in rat striatal membranes

Life Sciences, Vol. 39, pp. 2429-2434 Printed in the U.S.A.

Pergamon Journals

INFLUENCE OF PERTUSSIS TOXIN ON THE EFFECTS OF GUANINE NUCLEOTIDE ON ADENYLATE CYCLASE IN RAT STRIATAL MEMBRANES T. Arima I, T. Segawa 1 and Y. Nomura2

1Department o f Pharmacology I n s t i t u t e o f Pharmaceutical Sciences Hiroshima University School of Medicine, Kasumi 132-3 , Minami-ku, Hiroshima 734, Japan Department of Pharmacology Research Institute for WAKAN-YAKU (Oriental Medicine) Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 930-01, Japan (Received in final form September 16, 1986) Summary The influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase activity were investigated in rat striatal membranes. GTP promoted and inhibited the activity at 1 and I00 pM, respectively. The inhibitory effects of GTP were abolished by pretreatment of the membranes with pertussis toxin. GppNHp (guanyl5'-yl-B,y-imidodiphosphate) exerted only stimulatory effects and pertussis toxin did not affect the effects of GppNHp. GDP at i0 and i00 ~M caused significant inhibition which was completely suppressed by pertussis toxin. It is suggested that guanine nucleotide regulates the affinity of as in stimulatory GTP-binding regulatory protein to either 8y or catalytic units of adenylate cyclase in a flip-flop manner. Inhibitory GTP-binding regulatory protein seems to play a regulatory role in inhibiting as activity supplying the By heterodimer. It is known that adenylate cyclase activity is dually regulated by stimulatory (Ns) and inhibitory guanine nucleotide-binding regulatory protein (Ni) in such a manner that interaction occurs between the subonits, as, 8 and y in Ns and ~i, B and y in Ni (I). ~i itself possesses inhibitory effects on adenylate cyclase at relatively higher concentrations but exerts stimulatory effects at lower concentrations (I). In contrast, a By heterodimer, a putatively common dimer in Ns and Ni (2), does not directly interact with catalytic units of cyclase but has an inhibitory effects at lower concentrations (1,3), suggesting that main inhibition of the catalytic units by Ni is due to a mechanism by which By is transferred from Ni to as in Ns, forming ~sSy heterodimers° Dopamine-I (D-I) receptor- Ns and D-2 receptor- Ni coupling systems exist in striatal membranes of rats and cause stimulatory and inhibitory influences on adenylate cyclase activity, respectively (4,5). To identify the molecular mechanism of the interaction of as, ai, BY in Ns and Ni involved in regulation of adenylate cyclase in neuronal membranes, the influences of pertussis toxin (IAP) on the effects of GTP, GppNHp and GDP on adenylate cyclase were investigated in striatal membranes. * To whom correspondence should be addressed. 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Journals Ltd.

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Methods Striatal membranes. Wistar rats were used. Striatal membrane were prepared by the method of Usdin et alo (6). Briefly, after decapitation, the striatal tissues were minced and homogenized in 50 mM Tris-HCl buffer (pH 7.4) with a Polytron (setting No.6) for 30 sec and the homogenates were centrifuged twice at 50,000 x g for l0 min with resuspension of intermediate pellets in fresh buffer. Resultant pellets resuspended in 40 mM Tris-lIC1 buffer (pH 7.4) were used for adenylate cyclase assay. Pertussis toxin (IAP) treatment. Pretreatment of membranes with IAP was carried out by the modified methods of Nomura et al. (7). For preactivation of IAP, 50 pM of 1 mg/ml IAP in 0.I M sodium, potassium phosphate buffer (pH 7.4) containing 2 M urea and 50 ul of 40 mM dithiothreitol in 40 mM Tris-HCl buffer (pH 7.4) were mixed and incubated at 37°C for 15 rain. Then 1.9 ml of membrane suspension in 40 mM Tris-HCl buffer (pH 7.4) containing 4 mM MgCI_, l mM ATP, 10 mM thymidine, 2 mM EDTA, 0.i mM GTP and 1 mM NAD was added andZfurther incubated at 37°C for 30 min. Membranes were washed three times with ice-cold Tris-HCl buffer (pH 7°4)° As a control, 0.1 M sodium, potassium phosohate buffer (pH 7.4) without IAP was used. Adenylate cyclase assay. Adenylate cyclase activity was measured by the modified method of Salomon et al. (8) o The total volume of the reaction mixture of 400 pl contained 40 mM Tris-HC1 buffer (pH 7.4), 4 mM M g C l ~ 1 mM DTT, 0.6 mM EGTA, 1 mM cyclic AMP, 0.4 mM ATP containing 1.2 ~iCi of L [3H]ATP, the test drug or the vehicle and the membrane suspension. Membrane suspensions (0.4 to 0.5 mg protein) were added to the reaction mixture and preincubated at 4°C for 20 min. After the addition of ATP as a substrate, the reaction mixture was incubated at 30~C for I0 min. The reactions were terminated by adding 0.4 ml of 1 mM cyclic AMP and boiling for 3 min, the reaction mixture was subsequently centrifuged at 15,000 x g for 3 min and the supernatant (0.6 ml) was applied to a column of Dowex 50W x 4 (0.6 x 3 cm, +H form, 200 to 400 mesh) The initial 0.6 ml effluent plus io0 ml distilled water was discarded and the subsequent 2.0 ml water effluent was applied to the next column, alumina (0.7 x 3 cm, 70 to 230 mesh). The initial effluent plus 1.0 ml of SO mM Tris-HC1 buffer (pH 7.7) were discarded, the subsequent 3.0 ml buffer effluents were collected and the radioactivity was measured by a liquid scintillation spectrometer (Packard model 3325) with I0 ml of Bray's solution. Protein content was determined by the method of Lowry et al. (9). Statistical significances between control and test values were analyzed by Student's t-test. Results Basal activity of adenylate cyclase of striatal membranes was 547~0 + 57.2 pmol cyclic AMP formed / mg protein / rain. The activity tended to be increased (670 + 18.1 pmol/mg/min) by 25 ~g/ml IAP. GTP at 1 uM significantly (P<0.05) increased the activity but decreased it at I00 ~M (P<0oOl) (Fig. l-A). IAP completely abolished the inhibitory effects of lO0 ~M GTP. GppNHp at concentrations of 1 to i00 ~M caused a significant (P<0.01) activation which was not affected by IAP (Fig. l-B). GDP at i ~M did not affect the activity (Fig. 2-A) but significantly inhibited it at i0 (P<0o01) and i00 ~M (P<0.001). Pretreatment of membranes with IAP resulted in a suppression of GDP (i0 and i00 ~M)-induced inhibition° In the presence of 1 ~M GppNHp, the influences of GDP addition were examined and the results are shown in Fig. 2-B. GDP at 1 ~l caused significant (P<0.05) inhibition. The inhibition occurred in a concentration-dependent manner and the inhibition by i00 pM was lower than that in the absence of GppNHp. IAP caused a

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C-protein - Adenylate Cyclase in Striatum

w oJ

+

+÷

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4

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9o

o

% u

80

[ 6 -

6

1 o g [ G T P ( M ) ]

-1

og

5

4

[GppNHp(M)

]

FIG. I Influences of pertussis toxin (IAP) on the effects of GTP (Fig. I-A) and GppNHp (Fig. I-B) on adenylate cyclase activity in striatal membranes. Each value shows the mean + SE of three independent experiments.() O, Control;O ........O, TAP treatment. Significance; * P<0.05, ** p<0.01 vs. guanine nucleotide-free; + P<0.05, ++ P<0.001 vs. control.

÷+

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,

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FIG. 2 Influences of pertussis toxin (IAP) on the effects of GDP on adenylate cyclase activity of striatal membranes in the absence (Fig. 2-A) or presence (Fig° 2-B) of I ~M GppNHp. Each value shows the mean + SE of three independent experiments.() O, Control;Q .......Q, I A P treatment. Significance; * P
243]

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significant conversion of GDP effect from inhibition to activation. GDP at 1 pM, in particular, caused significant (P40.05) activation in IAP-pretreated membranes compared to that in control membranes. Discussion To gain an insight into the mechanism of the interaction of Ns- and Niregulating adenylate cyclase activity, the influences of IAP on the effects of guanine nucleotide on adenylate cyclase activity were examined in rat striatal membranes in which both Ns and Ni coexisted. as, By, GTP and GDP could interact with one another as shown in Fig. 3 (I).

GTPase as

(a) a? GDP

GDP ~

GTP \

J

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8¥

,-...

/By

~y ~/ (b)

(d)

L

as °B~" GTP

X(Ni) GTP

GTP a.SoBy GDP

. GDP ~ " x (c)

GTP FIG. 3

Proposed mechanism by Katada et al. (i) of the interaction between ~s, By and guanine nucleotideo ~s, ~ subunits of Ns; ai, ~ subunits of Ni. The activation of adenylate cyclase by 1 ~M GTP can probably be explained By preferential progress of reactions (c) and (d) (formation of ~s. GTP.67 and of as.GTP), although GTP may act on Ni and supply 67 from Ni to ~s (acct~nulation of as.GDP.By by the reaction (b))o GTP at lO0 }JM inhibited this activity. Supply of By from Ni, which accelerates the formation of asBy, underlies the inhibitory effects of GTP at high concentration. This is supported by the fact that IAP abolished the inhibitory effects of I00 pM GTP, since IAP is known to inhibit dissociation of ~i from Ni by ADP-ribosylation of ai (10,11). GppN~Ip activated the cyclase without any inhibition and IAP had no effect at all, suggesting that GppNHp may not act on Ni. It is difficult, however, to prestnne that GppNHp takes part in reaction (a) because of the fact that GTPase cannot hydrolyze GppNqp. Even though BY dimers are supplied from Xi, ~s. GppNlp could not bind By (namely the reaction (b) could not proceed). Therefore, the lack of inhibitory effects of GppNHp on adenylate cyclase is due to difficulty in formation of ~soGppNHp. By complex (lower affinity of ~s.GppNHp to By than that of ~s. GDP) rather than to insensitivity of Ni to GppNHp. In fact, it has been suggested that Ni is sensitive to GppNHp (12,13,14). GDP inhibited adenylate cyclase, probably through GDP-induced acceleration of reaction (b) and/or the inverse reaction of (c) (i.e., accelerated formation of ~s. GDP.By). Suppressive effects of IAP on GDP inhibition on adenylate

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C-protein - Adenylate Cyclase in Striatum

2433

cyclase seem to be due to the inhibitory effects of IAP on the formation of By supply (reaction (b)). On the other hand, in spite of IAP-induced acctnnulation of ~s.GDP followed by reduction of ~s.GDP.By, cyclase activity was not reduced, suggesting that ~soGDP may possess stimulatory effects or may not possess an effect on catalytic units° The fact that agonist stimulation of stimulatory receptors or A I F dissociates Ns into as and BY and then activates catalytic unlts even in the absence of GTP (15) may suggest a stimulatory function of ~s. GDP. Thus ~s.GDP could be formed by the inverse reaction of (b) and activate catalytic units. •

.

4

In the presence of 1 ~M GppNHp, 1 ]JM GDP inhibited cyclase in contrast to the lack of any inhibitory action of GDP in the absence of GppNHp. Formation of ~s.GDP by the addition of GDP could easily proceed to the formation of ~s°GDP by s u p p l y of 87 from Ni, since GppNHp dissociates Ni into ~i and By° This is well consistent with the idea that Ni is also sensitive to GppNHp, as described above. The lower inhibitory effects of I00 ~M GDP in the presence of GppNHp than that in the absence of GppNHp may be due to : I) the reduction of BY dimers (the reduction of ~s.GDP.By by dilution of GppNHp with high concentration of GDP) and 2) stimulatory effects of ~s. GppNHp on cyclase. In IAPpretreated membranes, 1 ~M GDP activated cyclase. GppNHp possibly accelerates reactions (c) and (d) but inhibits reaction (a). Thus ~s.GppNHp is increased, causing stimulation of cyclase. Moreover, it is presumable that the addition of 1 ~M GDP forms as.GDP but does not form ~s.GDP.By, since IAP inhibits the supply of By to ~s.GDP by ADP-ribosylation of ~i. Therefore, the total content of ~s. GppNHp and ~s.GDP could be increased, inducing an activation of cyclase. It is suggested that the interaction between Ns and Ni in the regulation of adenylate cyclase activity seems to be based on the transfer of By dimer between ~s and ~io Receptor stimulation or guanine nucleotide level modulates affinity of ~s and ~i to By. Thus a model of a regulatory mechanism of the flip-flop type with regard to ~s, BY and the catalytic units could be proposed as shown in Fig. 4. Activity of catalytic units could be reduced if GDP induced the formation of ~sBy. GTP, GppNHp, receptor stimulation or AIF 4- dissociates from as and then increases the affinity of ~s to the catalytic unit. Hydrolysis of GTP by GTPase or the addition of excess GDP results in the formation of ~soGDP which still activates the catalytic unit but in~nediately forms ~s.GDP.By if BY exists in the system. Thus guanine nucleotide plays regulatory roles in tile affinity of ~s to either By dimer or the catalytic nuit.

GDP

GTP

!

\

p

! C

\

By

GTPase, GDP, 8y

C*

By

FIG. 4 The flip-flop-type mechanism of the interaction between as, By and the catalytic unit in regulating adenylate cyclase activity. C, catalytic unit of adenylate cyclase; C*, active state of catalytic unit; Rs, stimulation of stimulatory receptor.

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Acknowledgements The authors are grateful to Prof. M. Ui of Hokkaido University and Drs. M. Yajima and K. Nogimori of Kaken Pharmaceutical Company for kind gift of IAP. This study was supported in part by Grants-in-Aid for Scientific Research from the Research Foundation for Pharmaceutical Sciences and from the Takeda Science Foundation in Japan. Re fe ten ces i. 2. 3. 4.

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