Neurochemical Research (1) 329-336 (1976)
E F F E C T OF R E S E R P I N E ON A D E N Y L A T E C Y C L A S E S Y S T E M OF RAT S T R I A T U M K. KUMAKURA, 1 A. CARENZI, 1 P. F. SPANO, 1 AND M. TRABUCCHI 2 1 Department of Pharmacology and Pharmacognosy and 2 Department of Pharmacology University of Milan, Milan, Italy
Accepted M a r c h 22, 1976
W e h a v e investigated t h e properties of d o p a m i n e - d e p e n d e n t adenylate cyclase in rat striatal h o m o g e n a t e s 20 h after reserpine treatment. In this experimental condition, we h a v e found that the affinity o f the e n z y m e for dopamine is greatly e n h a n c e d . On the other h a n d , the concentration of a p o m o r p h i n e required to produced half-maximal activation o f the e n z y m e in striatal h o m o g e n a t e s of controls and reserpine-treated rats is not changed. T h e u n c h a n g e d affinity of adenylate cyclase for the substrate ( A T P : Mg ++) indicates that reserpine probably affects the receptor c o m p o n e n t of the e n z y m e .
INTRODUCTION Much pharmacological evidence suggests that surgical or chemical denervation in the peripheral (1) and central nervous system (2-4) produces hypersensitivity to the exogenously applied neurotransmitter. Assuming that adenylate cyclase in some tissues is a part of the postsynaptic receptor for catecholamines, Weiss and Costa (5) found that norepinephrine (NE)-dependent adenylate cyclase, in chronically denervated rat pineal gland, is more responsive to the stimulatory effect of the transmitter when compared to the activity of the enzyme in innervated glands. A large body of evidence suggests that dopamine (DA)-dependent adenylate cyclase may be related to the dopaminergic receptor (6). Therefore, degeneration of presynaptic terminals would be expected to increase the sensitivity of DA-dependent adenylate cyclase to DA itself. 329 9 1976 Plenum PublishingCorporation, 227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming,recording,or otherwise, without written permissionof the publisher.
330
KUMAKURA ET AL.
It has been shown (7) that unilateral radiofrequency lesions or chemical lesions with 6 - O H D A in the substantia nigra of the rat resulted in a selective increase of the adenylate cyclase response to D A in striatal homogenates. On the other hand, Von Voightlander et al. (8) failed to demonstrate in mice treated with a-methyltyrosine for 13 days any supersensitivity of the e n z y m e to the neurotransmitter. ( + ) - A m p h e t a m i n e in reserpine-treated rats induces an increase of spontaneous m o t o r activity and aggression more evident than in control rats (Kumakura, unpublished observation). Carenzi et al. (9) and Chiueh and Moore (10) have suggested that ( + ) - a m p h e t a m i n e in reserpinetreated rats can release the newly synthesized D A from nigrostriatal neurons. H o w e v e r , this observation may be n o t s u f f i c i e n t to explain the enhanced behavior after (+)-amphetamine in reserpinized rats, since this p h e n o m e n o n can also be related to a supersensitivity of the dopaminergic receptor. Therefore, we decided to further investigate this problem, measuring the D A - d e p e n d e n t adenylate cyclase activity in rat striatal h o m o g e n a t e s 20 h after reserpine treatment.
EXPERIMENTAL
PROCEDURE
Sprague-Dawley rats (125-150 g) were sacrificed by decapitation. The brain was quickly removed, and the striata were rapidly dissected. Tissue samples were gently homogenized at 4~ in 25 vol 0.8 M tris-maleate buffer, pH 7.5. Adenylate cyclase activity was measured as described by Carenzi et al. (11), using 8-14C adenosine triphosphate (ATP) as substrate. The final incubation mixture of 500 txl contained 0.8 M tris-maleate buffer, pH 7.5; 0.5 mM EGTA; 2 mM MgSO4; 10 mM theophylline; 1 mM 14C-ATP(0.98 mCi/mM); and 180-150/xg tissue proteins. After incubation for 4 min at 30~ the reaction was stopped by boiling the tubes for 3 min. Radioactive 14C-cyclic 3'5'-adenosinemonophosphate (cAMP) was separated from radioactive ATP using aluminum and Dowex columns, as described by Guidotti and Costa (12). Protein was measured by the method of Lowry et al. (13). ATP-~4Cwas obtained from the Radiochemical Centre, Amersham. Rats were treated by intrapefitoneal injection of 6 mg/kg od reserpine (HCI salt) 20 h before the sacrifice.
RESULTS Adenylate cyclase activity has been measured in striatal homogenates of reserpine-treated rats in the presence of D A or apomorphine (Table I). The basal activity of the e n z y m e was found to be u n c h a n g e d in
RESERPINE- AND DOPAMINE-DEPENDENT
ADENYLATE
CYCLASE
331
TABLE I EFFECT OF DOPAMINE AND APOMORPHINE ON ADENYLATE CYCLASE ACTIVITY IN STRIATAL HOMOGENATES OF SALINE- AND RESERPINE-TREATED RATS a c A M P formed (pmol)/min per mg protein Addition to assay
Saline-treated
None Dopamine, 5 • 10-6 M Dopamine, 5 • 10-5 M Apomorphine, 5 • 10-6 M Apomorphine, 5 • 10-~ M
306.6 545.7 816.5 420.8 490.2
-+ 15(0 -4- 36.1 -+ 34.8 -+ 25.5 + 30.7
Reserpine-treated 292.8 653.8 1104.3 565.1 599.6
-+ 23.8 -+ 31.7 b -+ 52.3 c + 39.0 b -+ 40.8 b
Increase (%) Saline
Reserpine
86 179 44 67
114 260 84 95
a Animals were treated with saline or reserpine (6 me/ks) intraperitoneally 20 h before decapitation. Each value for the enzyme activity is an average --+SE of at least 5 experiments. b p < 0.005 when compared with the activity of adenylate cyclase in striatal homogenates of saline-treated rats in the presence of the same concentration of D A or apomorphine. e p < 0.001 when compared with the activity of adenylate cyclase in striatal homogenates of saline-treated rats in the presence of the same concentration of DA or apomorphine.
homogenates of saline- and reserpine-treated rats. Dopamine in concentrations of 5• -6 and 5• -5 M increased the adenylate cyclase activity in striatal homogenates of saline-treated rats by, respectively, 86 and 179%. The same concentrations of DA added to the homogenate s of reserpine-treated rats increased the enzyme activity by 114 and 260% of the basal activity, respectively. Apomorphine in concentrations of 5• -G and 5• 10-5 M increased the adenylate cyclase activity by 44 TABLE II EFFECT OF N a F AND N E ON ADENYLATE CYCLASE ACTIVITY IN STRIATAL HOMOGENATES OF SALINE- AND RESERPINE-TREATED RATS a c A M P formed (pmol)/min per mg protein Addition to assay
Saline-treated
Reserpine-treated
None N a F , 10 -2 M N E 5 • 10-6 M
259.3 -+ 11.3 367.0 -+ 24.1 b 352.8 _+ 38.4 b
252.4 + 14.6 348.1 + 21.7 ~ 347.6 + 42.4 b
The animals were treated with saline and reserpine (6 rag/ks) intraperitoneally. Each value is an average -+s~ of at least 4 experiments. b p < 0.05 when compared with the enzyme activity on striatal homogenates without addition of N a F and RE.
332
K U M A K U R A ET AL.
A c
"-: 0
0~04_
-5
09O3-
E
.~
0~02_
FIG. 1. Double-reciprocal plots of c A M P increase as a function of DA concentration in striatal homogenates of control (A) and reserpine-treated rats (B). The animals were treated as described in Table I. In the absence of added DA, the adenylate cyclase activity in striatal homogenates of control and reserpine-treated rats was, respectively, 302-+ 12.3 and 287-+ 16.8 pmol c A M P formed in 1 min of incubation/mg protein. Each point represents the mean of 3 different experiments conducted in triplicate.
and 67%, respectively, in the homogenates of saline-treated rats, and by 84 and 95% in the homogenates of reserpine-treated animals. Figure 1 shows the double-reciprocal plot of c A M P increase as a function of DA concentrations in striatal homogenates of control (A) and reserpine-treated (B) rats. The affinity of the enzyme for DA is increased almost 3-fold in the homogenates of reserpine-treated rats. Figure 2 shows the double-reciprocal plot of c A M P increase as a function of apomorphine concentrations in the striatal homogenates of control (A) and reserpine-treated (B) rats. No change in the apparent Km value was found, but the enzyme reaction velocity was higher in the striatal homogenates of reserpine-treated rats. Table II shows the stimulatory effects of N a F (10 -2 M) and norepinephrine (NE) (5• 10-6 M) on the adenylate cyclase activity in striatal homogenates of control and reserpinized rats. No difference between the two groups was found in the stimulating effects of N a F and NE. Figure 3 shows the effects of DA and apomorphine on the kinetic
RESERPINE- AND DOPAMINE-DEPENDENT
333
A D E N Y L A T E CYCLASE
properties of the enzyme in vitro. A 1:4 ratio of ATP:Mg ++ was maintained in this experiment, as described by Clement-Cormier et al. (14). The affinity of the enzyme for ATP was not changed by reserpine treatment; furthermore, neither DA nor apomorphine added in vitro had effect on the affinity of the enzyme for the substrate in the striatal homogenates of either saline- or reserpine-treated rats.
DISCUSSION Recently, Siggins et al. (15) reported an increased potency of iontophoretically applied DA and apomorphine on caudate neurons in animals with chemical lesions of the substantia nigra. After destruction of a nigrostriatal pathway, the systemic administration of different dopaminergic ag0nists produces an enhanced behavior that suggests a receptor supersensitivity (2). The similarity between DA
.g
0.012
o_
0.009] ~ o~o6 '
-0.5
-04
-0.3
-0.2
-0.I
o!1
A
o!2
o!3
o!4
o!5
I/Apomorphine (~ M) FI6. 2. Double-reciprocal plots of c A M P increase as a function of apomorphine concentrations in striatal homogenate of control (A) and reserpine-treated rats (B). The animals were treated as described in Table I. In the absence of added apomorphine, the adenylate cyclase activity in striatal homogenates of control and reserpine-treated rats was, respectively, 319+17.6 and 303-+21.8 pmol c A M P formed in 1 rain of incubation/mg protein. Each point represents the mean of 3 different experiments conducted in triplicate.
334
K U M A K U R A ET AL.
Control 0.015
E Apo
/
/ /
E "o
//
E
9
/ Apo
,/
/
v,.
D.
DA
DA
//t
< O
m O
E O,. v
-0" ~ ///I
J A r P] ~[M g'l
01,
0!~
0!,,
IJATP
_
4
(lO-' M )
f,.0
FIG. 3. Double-reciprocal plots of c A M P formation as a function of ATP concentration in striatal homogenate of saline- ( 0 - 0 ) and reserpine-treated (A-A) rats, in the absence or presence of dopamine (DA) (5 /xM) and of apomorphine (Apo) (5 /xM). A 4:1 ratio of Mg++: ATP was maintained. The animals were treated as described in Table I.
RESERPINE- A N D D O P A M I N E - D E P E N D E N T A D E N Y L A T E CYCLASE
335
receptor and the DA-dependent adenylate cyclase (6) and the increased sensitivity of this enzyme to DA after chronic denervation (7) provide indirect evidence for an important role of this enzyme in the transmitter function of DA, and indicate that the change of DA receptor occurring after denervation implies a change of the enzyme activity. Our results provide evidence that the affinity for DA of adenyiate cyclase in striatal homogenates of reserpinized rats is increased (Fig. 1). The apparent Km value for DA in reserpinized rats is equal to that for apomorphine, which was not changed by reserpine treatment (Fig. 2). This result suggests that the affinity of the receptor for apomorphine is already maximized. The velocity of the enzyme stimulated by apomorphine is 2-3-fold lower than that of the enzyme stimulated by DA, suggesting that not all the receptors sensitive to DA are also sensitive to apomorphine. Since Snyder and Coyle (16) have shown that the reuptake of DA in striatal homogenate was not affected by reserpine treatment, our results may not depend on changes of the reuptake system. The unchanged affinity of adenylate cyclase for the substrate (ATP: Mg § indicates that reserpine treatment probably results in a change of the receptor component of the enzyme. The reasons for the increased affinity of the receptor for DA might be: (a) Reserpine reduces the presence of the socalled spare receptors (17), mainly inactive ones; thus, less DA is enough to obtain the maximal activity; (b) reserpine may produce some changes in the level of the receptor part and may also affect the catalytic component (18), with or without preexisting receptor excess. That there was no supersensitivity for N a F and NE supports the hypothesis of a relationship between dopamine supersensitivity and the dopamine-stimulated component of the striatal cyclase.
REFERENCES 1. BRIM1JOIN, S., PLUCHINO, S., and TRENDELEMBURG,U. (1970) On the mechanism of supersensitivity to norepinephrine in the denervated cat spleen. J. Pharmacol. Exp. Ther. 175,503-513. 2. UNGERSTEDT, U. (1971) Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigrostriatal dopamine system. Acta Physiol. Scand. Suppl. 367, 69-93. 3. TARSY, S., and BALDESSARINI, R. J. (1974) Behavioral supersensitivity to apomorphine following chronic treatment with drugs which interfere with the synaptic function of catecholamines. Neuropharmacology 13,927-940. 4. GIANUTSOS, G., DRAWBAUGH, R. B., HYNES, M. D., and LAL, H. (1974) Behavioral
336
5. 6.
7.
8.
9.
10.
11.
12. 13. 14.
15.
16.
17. 18.
K U M A K U R A ET AL.
evidence for dopaminergic supersensitivity after chronic haloperidol. Life Sci. 14, 887898. WEISS,B., and COSTA, E. (1967) Adenyl cyclase activity in rat pineal gland: effects of chronic denervation and norepinephrine. Science 156, 1750-1752. KEBABIAN, J. N., PETZOLD, G. L., and GREENGARD, P. (1972) Dopamine sensitive adenylate cyclase in caudate nucleus of rat brain and its similarity to the "dopamine receptor." Proc. Nat. Acad. Sci. U.S.A. 69, 2145-2149. MISHRA, R. K., GARDNER, E. L., KATZMAN, R., and MAKMAN, M. H. (1974) Enhancement of dopamine stimulated adenylate cyclase activity in rat caudate after lesions in substantia nigra: evidence for denervation supersensitivity. Proc. Nat. Acad. Sci. U.S.A. 71, 3883-3887. VON VOIGHTLANDER, P. F., BOUKMA, S. T., and JOHNSON, G. A. (1973) Dopaminergic denervation supersensitivity and dopamine stimulated adenyl cyclase activity. Neuropharmacology 12, 1081-1086. CARENZI, A., CHENEY, D. L., COSTA, E., GUIDOTTI, A., and RACAGNI, G. (1975) Action of opiates, antipsych0tics, amphetamine and apomorphine on dopamine receptors in rat striatum: in vivo changes of Y-5'-cyclic adenosine monophosphate content and acetylcholine turnover rate. Neuropharmacology (in press). CHIUEH, C. C., and MOORE, K. E. (1975) D-Amphetamine-induced release of "newly synthesized" and "stored" dopamine from the caudate nucleus in vivo. J. Pharmacol. Exp. Ther. 192, 642-653. CARENZI, A., GILLIN, G., GUIDOTTI, A., SCHWARTZ, M., TRABUCCHI, M., and WYATT, R. (1975) Dopamine-sensitive adenyl cyclase in human caudate nucleus: a study in control subjects and schizophrenic patients. Arch. Gen. Psychiatry 32, 10561059. GUIDOTTI,A., and COSTA, E. (1974) A role of nicotinic receptors in the regulation of the adenylate cyclase of adrenal medulla. J. Pharmacol. Exp. Ther. 189, 665-675. LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,265-275. CLEMENT-CORMIER,Y. C., PARRISH, R. G., PETZOLD, G. L., KEBABIAN, J. W., and GREENGARD, P. (1975) Characterization of a dopamine-sensitive adenylate cyclase in the rat caudate nucleus. J. Neurochem. 25, 143-149. SIGGINS, G. R., HOFFER, B. J., and UNGERSTEDT, U. (1974) Electrophysiological evidence for involvement of cyclic adenosine monophosphate in dopamine responses of caudate neurons. Life Sci. 15,779-792. SNYDER, S. H., and COYLE, J. T. (1969) Regional differences in Ha-norepinephrine and H3-dopamine uptake in rat brain homogenates. J. Pharmacol. Exp. Ther. 165, 7886. STEPHENSON, R. P. (1956) A modification of receptor theory. Br. J. Pharmacol. 11, 379-393. PERKINS, J. P. (1973) Adenyl cyclase, in GREENGARD,P., and ROBINSON, G. A. (eds.), Advances in Cyclic Nucleotide Research, Vol. 3, Raven Press, New York, pp. 11-16.
E F F E C T OF R E S E R P I N E ON A D E N Y L A T E C Y C L A S E S Y S T E M OF RAT S T R I A T U M K. KUMAKURA, 1 A. CARENZI, 1 P. F. SPANO, 1 AND M. TRABUCCHI 2 1 Department of Pharmacology and Pharmacognosy and 2 Department of Pharmacology University of Milan, Milan, Italy
Accepted M a r c h 22, 1976
W e h a v e investigated t h e properties of d o p a m i n e - d e p e n d e n t adenylate cyclase in rat striatal h o m o g e n a t e s 20 h after reserpine treatment. In this experimental condition, we h a v e found that the affinity o f the e n z y m e for dopamine is greatly e n h a n c e d . On the other h a n d , the concentration of a p o m o r p h i n e required to produced half-maximal activation o f the e n z y m e in striatal h o m o g e n a t e s of controls and reserpine-treated rats is not changed. T h e u n c h a n g e d affinity of adenylate cyclase for the substrate ( A T P : Mg ++) indicates that reserpine probably affects the receptor c o m p o n e n t of the e n z y m e .
INTRODUCTION Much pharmacological evidence suggests that surgical or chemical denervation in the peripheral (1) and central nervous system (2-4) produces hypersensitivity to the exogenously applied neurotransmitter. Assuming that adenylate cyclase in some tissues is a part of the postsynaptic receptor for catecholamines, Weiss and Costa (5) found that norepinephrine (NE)-dependent adenylate cyclase, in chronically denervated rat pineal gland, is more responsive to the stimulatory effect of the transmitter when compared to the activity of the enzyme in innervated glands. A large body of evidence suggests that dopamine (DA)-dependent adenylate cyclase may be related to the dopaminergic receptor (6). Therefore, degeneration of presynaptic terminals would be expected to increase the sensitivity of DA-dependent adenylate cyclase to DA itself. 329 9 1976 Plenum PublishingCorporation, 227 West 17th Street, New York, N.Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming,recording,or otherwise, without written permissionof the publisher.
330
KUMAKURA ET AL.
It has been shown (7) that unilateral radiofrequency lesions or chemical lesions with 6 - O H D A in the substantia nigra of the rat resulted in a selective increase of the adenylate cyclase response to D A in striatal homogenates. On the other hand, Von Voightlander et al. (8) failed to demonstrate in mice treated with a-methyltyrosine for 13 days any supersensitivity of the e n z y m e to the neurotransmitter. ( + ) - A m p h e t a m i n e in reserpine-treated rats induces an increase of spontaneous m o t o r activity and aggression more evident than in control rats (Kumakura, unpublished observation). Carenzi et al. (9) and Chiueh and Moore (10) have suggested that ( + ) - a m p h e t a m i n e in reserpinetreated rats can release the newly synthesized D A from nigrostriatal neurons. H o w e v e r , this observation may be n o t s u f f i c i e n t to explain the enhanced behavior after (+)-amphetamine in reserpinized rats, since this p h e n o m e n o n can also be related to a supersensitivity of the dopaminergic receptor. Therefore, we decided to further investigate this problem, measuring the D A - d e p e n d e n t adenylate cyclase activity in rat striatal h o m o g e n a t e s 20 h after reserpine treatment.
EXPERIMENTAL
PROCEDURE
Sprague-Dawley rats (125-150 g) were sacrificed by decapitation. The brain was quickly removed, and the striata were rapidly dissected. Tissue samples were gently homogenized at 4~ in 25 vol 0.8 M tris-maleate buffer, pH 7.5. Adenylate cyclase activity was measured as described by Carenzi et al. (11), using 8-14C adenosine triphosphate (ATP) as substrate. The final incubation mixture of 500 txl contained 0.8 M tris-maleate buffer, pH 7.5; 0.5 mM EGTA; 2 mM MgSO4; 10 mM theophylline; 1 mM 14C-ATP(0.98 mCi/mM); and 180-150/xg tissue proteins. After incubation for 4 min at 30~ the reaction was stopped by boiling the tubes for 3 min. Radioactive 14C-cyclic 3'5'-adenosinemonophosphate (cAMP) was separated from radioactive ATP using aluminum and Dowex columns, as described by Guidotti and Costa (12). Protein was measured by the method of Lowry et al. (13). ATP-~4Cwas obtained from the Radiochemical Centre, Amersham. Rats were treated by intrapefitoneal injection of 6 mg/kg od reserpine (HCI salt) 20 h before the sacrifice.
RESULTS Adenylate cyclase activity has been measured in striatal homogenates of reserpine-treated rats in the presence of D A or apomorphine (Table I). The basal activity of the e n z y m e was found to be u n c h a n g e d in
RESERPINE- AND DOPAMINE-DEPENDENT
ADENYLATE
CYCLASE
331
TABLE I EFFECT OF DOPAMINE AND APOMORPHINE ON ADENYLATE CYCLASE ACTIVITY IN STRIATAL HOMOGENATES OF SALINE- AND RESERPINE-TREATED RATS a c A M P formed (pmol)/min per mg protein Addition to assay
Saline-treated
None Dopamine, 5 • 10-6 M Dopamine, 5 • 10-5 M Apomorphine, 5 • 10-6 M Apomorphine, 5 • 10-~ M
306.6 545.7 816.5 420.8 490.2
-+ 15(0 -4- 36.1 -+ 34.8 -+ 25.5 + 30.7
Reserpine-treated 292.8 653.8 1104.3 565.1 599.6
-+ 23.8 -+ 31.7 b -+ 52.3 c + 39.0 b -+ 40.8 b
Increase (%) Saline
Reserpine
86 179 44 67
114 260 84 95
a Animals were treated with saline or reserpine (6 me/ks) intraperitoneally 20 h before decapitation. Each value for the enzyme activity is an average --+SE of at least 5 experiments. b p < 0.005 when compared with the activity of adenylate cyclase in striatal homogenates of saline-treated rats in the presence of the same concentration of D A or apomorphine. e p < 0.001 when compared with the activity of adenylate cyclase in striatal homogenates of saline-treated rats in the presence of the same concentration of DA or apomorphine.
homogenates of saline- and reserpine-treated rats. Dopamine in concentrations of 5• -6 and 5• -5 M increased the adenylate cyclase activity in striatal homogenates of saline-treated rats by, respectively, 86 and 179%. The same concentrations of DA added to the homogenate s of reserpine-treated rats increased the enzyme activity by 114 and 260% of the basal activity, respectively. Apomorphine in concentrations of 5• -G and 5• 10-5 M increased the adenylate cyclase activity by 44 TABLE II EFFECT OF N a F AND N E ON ADENYLATE CYCLASE ACTIVITY IN STRIATAL HOMOGENATES OF SALINE- AND RESERPINE-TREATED RATS a c A M P formed (pmol)/min per mg protein Addition to assay
Saline-treated
Reserpine-treated
None N a F , 10 -2 M N E 5 • 10-6 M
259.3 -+ 11.3 367.0 -+ 24.1 b 352.8 _+ 38.4 b
252.4 + 14.6 348.1 + 21.7 ~ 347.6 + 42.4 b
The animals were treated with saline and reserpine (6 rag/ks) intraperitoneally. Each value is an average -+s~ of at least 4 experiments. b p < 0.05 when compared with the enzyme activity on striatal homogenates without addition of N a F and RE.
332
K U M A K U R A ET AL.
A c
"-: 0
0~04_
-5
09O3-
E
.~
0~02_
FIG. 1. Double-reciprocal plots of c A M P increase as a function of DA concentration in striatal homogenates of control (A) and reserpine-treated rats (B). The animals were treated as described in Table I. In the absence of added DA, the adenylate cyclase activity in striatal homogenates of control and reserpine-treated rats was, respectively, 302-+ 12.3 and 287-+ 16.8 pmol c A M P formed in 1 min of incubation/mg protein. Each point represents the mean of 3 different experiments conducted in triplicate.
and 67%, respectively, in the homogenates of saline-treated rats, and by 84 and 95% in the homogenates of reserpine-treated animals. Figure 1 shows the double-reciprocal plot of c A M P increase as a function of DA concentrations in striatal homogenates of control (A) and reserpine-treated (B) rats. The affinity of the enzyme for DA is increased almost 3-fold in the homogenates of reserpine-treated rats. Figure 2 shows the double-reciprocal plot of c A M P increase as a function of apomorphine concentrations in the striatal homogenates of control (A) and reserpine-treated (B) rats. No change in the apparent Km value was found, but the enzyme reaction velocity was higher in the striatal homogenates of reserpine-treated rats. Table II shows the stimulatory effects of N a F (10 -2 M) and norepinephrine (NE) (5• 10-6 M) on the adenylate cyclase activity in striatal homogenates of control and reserpinized rats. No difference between the two groups was found in the stimulating effects of N a F and NE. Figure 3 shows the effects of DA and apomorphine on the kinetic
RESERPINE- AND DOPAMINE-DEPENDENT
333
A D E N Y L A T E CYCLASE
properties of the enzyme in vitro. A 1:4 ratio of ATP:Mg ++ was maintained in this experiment, as described by Clement-Cormier et al. (14). The affinity of the enzyme for ATP was not changed by reserpine treatment; furthermore, neither DA nor apomorphine added in vitro had effect on the affinity of the enzyme for the substrate in the striatal homogenates of either saline- or reserpine-treated rats.
DISCUSSION Recently, Siggins et al. (15) reported an increased potency of iontophoretically applied DA and apomorphine on caudate neurons in animals with chemical lesions of the substantia nigra. After destruction of a nigrostriatal pathway, the systemic administration of different dopaminergic ag0nists produces an enhanced behavior that suggests a receptor supersensitivity (2). The similarity between DA
.g
0.012
o_
0.009] ~ o~o6 '
-0.5
-04
-0.3
-0.2
-0.I
o!1
A
o!2
o!3
o!4
o!5
I/Apomorphine (~ M) FI6. 2. Double-reciprocal plots of c A M P increase as a function of apomorphine concentrations in striatal homogenate of control (A) and reserpine-treated rats (B). The animals were treated as described in Table I. In the absence of added apomorphine, the adenylate cyclase activity in striatal homogenates of control and reserpine-treated rats was, respectively, 319+17.6 and 303-+21.8 pmol c A M P formed in 1 rain of incubation/mg protein. Each point represents the mean of 3 different experiments conducted in triplicate.
334
K U M A K U R A ET AL.
Control 0.015
E Apo
/
/ /
E "o
//
E
9
/ Apo
,/
/
v,.
D.
DA
DA
//t
< O
m O
E O,. v
-0" ~ ///I
J A r P] ~[M g'l
01,
0!~
0!,,
IJATP
_
4
(lO-' M )
f,.0
FIG. 3. Double-reciprocal plots of c A M P formation as a function of ATP concentration in striatal homogenate of saline- ( 0 - 0 ) and reserpine-treated (A-A) rats, in the absence or presence of dopamine (DA) (5 /xM) and of apomorphine (Apo) (5 /xM). A 4:1 ratio of Mg++: ATP was maintained. The animals were treated as described in Table I.
RESERPINE- A N D D O P A M I N E - D E P E N D E N T A D E N Y L A T E CYCLASE
335
receptor and the DA-dependent adenylate cyclase (6) and the increased sensitivity of this enzyme to DA after chronic denervation (7) provide indirect evidence for an important role of this enzyme in the transmitter function of DA, and indicate that the change of DA receptor occurring after denervation implies a change of the enzyme activity. Our results provide evidence that the affinity for DA of adenyiate cyclase in striatal homogenates of reserpinized rats is increased (Fig. 1). The apparent Km value for DA in reserpinized rats is equal to that for apomorphine, which was not changed by reserpine treatment (Fig. 2). This result suggests that the affinity of the receptor for apomorphine is already maximized. The velocity of the enzyme stimulated by apomorphine is 2-3-fold lower than that of the enzyme stimulated by DA, suggesting that not all the receptors sensitive to DA are also sensitive to apomorphine. Since Snyder and Coyle (16) have shown that the reuptake of DA in striatal homogenate was not affected by reserpine treatment, our results may not depend on changes of the reuptake system. The unchanged affinity of adenylate cyclase for the substrate (ATP: Mg § indicates that reserpine treatment probably results in a change of the receptor component of the enzyme. The reasons for the increased affinity of the receptor for DA might be: (a) Reserpine reduces the presence of the socalled spare receptors (17), mainly inactive ones; thus, less DA is enough to obtain the maximal activity; (b) reserpine may produce some changes in the level of the receptor part and may also affect the catalytic component (18), with or without preexisting receptor excess. That there was no supersensitivity for N a F and NE supports the hypothesis of a relationship between dopamine supersensitivity and the dopamine-stimulated component of the striatal cyclase.
REFERENCES 1. BRIM1JOIN, S., PLUCHINO, S., and TRENDELEMBURG,U. (1970) On the mechanism of supersensitivity to norepinephrine in the denervated cat spleen. J. Pharmacol. Exp. Ther. 175,503-513. 2. UNGERSTEDT, U. (1971) Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigrostriatal dopamine system. Acta Physiol. Scand. Suppl. 367, 69-93. 3. TARSY, S., and BALDESSARINI, R. J. (1974) Behavioral supersensitivity to apomorphine following chronic treatment with drugs which interfere with the synaptic function of catecholamines. Neuropharmacology 13,927-940. 4. GIANUTSOS, G., DRAWBAUGH, R. B., HYNES, M. D., and LAL, H. (1974) Behavioral
336
5. 6.
7.
8.
9.
10.
11.
12. 13. 14.
15.
16.
17. 18.
K U M A K U R A ET AL.
evidence for dopaminergic supersensitivity after chronic haloperidol. Life Sci. 14, 887898. WEISS,B., and COSTA, E. (1967) Adenyl cyclase activity in rat pineal gland: effects of chronic denervation and norepinephrine. Science 156, 1750-1752. KEBABIAN, J. N., PETZOLD, G. L., and GREENGARD, P. (1972) Dopamine sensitive adenylate cyclase in caudate nucleus of rat brain and its similarity to the "dopamine receptor." Proc. Nat. Acad. Sci. U.S.A. 69, 2145-2149. MISHRA, R. K., GARDNER, E. L., KATZMAN, R., and MAKMAN, M. H. (1974) Enhancement of dopamine stimulated adenylate cyclase activity in rat caudate after lesions in substantia nigra: evidence for denervation supersensitivity. Proc. Nat. Acad. Sci. U.S.A. 71, 3883-3887. VON VOIGHTLANDER, P. F., BOUKMA, S. T., and JOHNSON, G. A. (1973) Dopaminergic denervation supersensitivity and dopamine stimulated adenyl cyclase activity. Neuropharmacology 12, 1081-1086. CARENZI, A., CHENEY, D. L., COSTA, E., GUIDOTTI, A., and RACAGNI, G. (1975) Action of opiates, antipsych0tics, amphetamine and apomorphine on dopamine receptors in rat striatum: in vivo changes of Y-5'-cyclic adenosine monophosphate content and acetylcholine turnover rate. Neuropharmacology (in press). CHIUEH, C. C., and MOORE, K. E. (1975) D-Amphetamine-induced release of "newly synthesized" and "stored" dopamine from the caudate nucleus in vivo. J. Pharmacol. Exp. Ther. 192, 642-653. CARENZI, A., GILLIN, G., GUIDOTTI, A., SCHWARTZ, M., TRABUCCHI, M., and WYATT, R. (1975) Dopamine-sensitive adenyl cyclase in human caudate nucleus: a study in control subjects and schizophrenic patients. Arch. Gen. Psychiatry 32, 10561059. GUIDOTTI,A., and COSTA, E. (1974) A role of nicotinic receptors in the regulation of the adenylate cyclase of adrenal medulla. J. Pharmacol. Exp. Ther. 189, 665-675. LOWRY, O. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193,265-275. CLEMENT-CORMIER,Y. C., PARRISH, R. G., PETZOLD, G. L., KEBABIAN, J. W., and GREENGARD, P. (1975) Characterization of a dopamine-sensitive adenylate cyclase in the rat caudate nucleus. J. Neurochem. 25, 143-149. SIGGINS, G. R., HOFFER, B. J., and UNGERSTEDT, U. (1974) Electrophysiological evidence for involvement of cyclic adenosine monophosphate in dopamine responses of caudate neurons. Life Sci. 15,779-792. SNYDER, S. H., and COYLE, J. T. (1969) Regional differences in Ha-norepinephrine and H3-dopamine uptake in rat brain homogenates. J. Pharmacol. Exp. Ther. 165, 7886. STEPHENSON, R. P. (1956) A modification of receptor theory. Br. J. Pharmacol. 11, 379-393. PERKINS, J. P. (1973) Adenyl cyclase, in GREENGARD,P., and ROBINSON, G. A. (eds.), Advances in Cyclic Nucleotide Research, Vol. 3, Raven Press, New York, pp. 11-16.
