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in vivo and in vitro evidence of dopaminergic system down regulation induced by chronic L-DOPA
Life Sciences, Vol. 34, pp. 2~07-2116 Printed in the U.S.A.
Pergamon Press
IN VIVO AND IN VITRO EVIDENCE OF DOPAMINERGIC SYSTEM DOWN REGULATION INDUCED BY CHRONIC L-DOPA
F. Ponzio, M. Cimino, G. Achilli, M. Lipartiti, C. Perego, G. Vantini and S. Alger~ Istituto di Ricerche Farmacologiche '~ario Negri" - via Eritrea, 62 20157 Milan, Italy. (Received in final form March 19, 1984) Summary The biochemical modifications which occur in the dopaminergic system after chronic administration of L-DOPA are investiga~e~ Levels of DA and of its metabolite 3-methoxytyramine (3-MT), an expression of the amount of DA released, were raised to the same extent in controls given a single dose of I-DOPA and in chronically treated rats given iOO mg/kg of I-DOPA plus 25 mg/kg of benserazide twice a day for 24 days. However, the reduction in neuronal function expressed by the decrease in 3-MT which follows treatment with DA agonists such as piribedil and apomorphine was less pronounced in the chronically L-DOPA treated rats. This suggests that such treatment causes a down regulation of DA receptors. These in vivo results were confirmed by in vitro analysis of DA receptor activity after chronic L-DOPA. Under these conditions there was a significant reduction in the number of L-3H_7-spiperone and L-3H_/-ADTN binding sites with no changes in their affinity. The in vivo and in vitro findings both suggest the involvement of a subsensitive compensatory mechanism or down regulation of dopaminergic neurons after chronic treatment with L-DOPA. Long-term levodopa (L-DOPA) therapy in Parkinson's disease is often associated with a decrease in the efficacy of this drug (1-4). The causes of this"tolerance" remain largely unclarified although on a theoretical basis the hypothesis can be formulated of a down-regulatory of some of the mechanisms of dopaminergic transmission, brought about by chronic dopaminergic hyperstimulation (5-8). In fact L-DOPA raises the concentration of dopamine (DA) at receptor sites (9) and therefore it might be considered an indirect dopaminergic agonist (iO). Experiments to date to test the possibility that chronic L-DOPA reduces the sensitivity of dopaminergic receptors have given conflicting results. Two levels of action should be considemed in tackling this question. One is at the presynapse: chronic hyperfunction could result in less release of DA formed from exogenous L-DOPA or it could affect tyrosine hydroxylase so that the activity of this enzyme remains inhibited beyond the period during which the concentration of DA is high. The other possible level of action of chronic L-DOPAis at the receptors: chronic hyperstimulation of DA receptors could in fact render them hyposensitive, thus explaining the decreased response to the neurotransmitter even when a high concentration is present. iCorrespondence
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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Dopaminergic Function After Chronic L-DOPA
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We have tested the two hypothesis in rats treated chronically with L-DOPA by determining i) whether the formation and release of DA after a single dose of L-DOPA and after chronic treatment is the same or not - DA release was evaluated by measuring changes in the concentration of 3-methoxy-4-hydroxytyramine (3-MT) ~, an intrasynaptic DA metabolite that reflects the amount of DA present in the synaptic cleft (9,11-13); 2) whether chronic treatment influences the activity of tyrosine hydroxylase (TH); 3) whether there is any change in DA receptor sensitivity. Our investigations included changes in specific binding of DA agonists and antagonists, and the efficiency of the negative feed-back response which usually follows stimulation of dopaminergic receptors by agonist drugs (9,14,15). Materials and Methods Animals CD-COBS male rats (Charles River, Italy) weighing 175 + 20 g were housed under standard conditions with free access to food and water. They were treated orally twice a day for 24 days with a suspension in 0.5% carboxymethylcellulose (C.M.C.) of i00 mg/kg or L-DOPA and 25 mg/kg of benserazide, an inhibitor of peripheral decarboxylase (16). Chronically L-DOPA + benserazide treated rats were given the following drugs as single doses, pyribedil, apomorphine and pargyline. Rats were killed 24 hours after last L-DOPA dose for determination of the activity of the enzyme TH and of DA and its metabolites, or 48 hafter the last L-DOPA dose for determining /-3H 7-spiperone or ~-3H_7-ADTN receptor binding. Rats were killed by d$cap~tation except when DA and its metabolites had to be assayed, in which case microwave irradiation (1.3 at 2.45 GHz for 4.25 sec) was used in order to rapidly inactivate catechol-o-methyl-transferase (COMT; E.C. 2.11.6) (17). In boyh cases striata were rapidly dissected, frozen on dry ice and kept at -80°C until biochemical assay.
a. DA and 3-MT For the simultaneous assay of 3-MT and DA the striatum of one animal was homogenized by sonifier in 300 ~i of 0.4N perchloric acid and centrifuged. The clear supernatant was adjusted to pH 7.5 - 7.6 to separate DA from 3-MT. At this pH DA was adsorbed onto alumina and 3-MT remained in the supernatant from which it was extracted according to our previously described methods (11,12). DA was eluted from the alumina according to Keller et al. (18). Liquid chromatography with electrochemical detection (LCEC) (Bioanalytical System Inc., West Lafayette, Ind.) was used for all biochemical assays. A glass column 500 n~n long, 2 mm i.d., packed with a cation exchange resin (Vydac CX, P310), The Separation Group Experia, CA) was used for DA determination. Samples were eluted with citrate/acetate buffer pH 5.2, 0.03 M; the detector was set at a potential of + 0.75 V. 3-MT was separated in a glass column 300 mm long, 2 mm i.d., packed with ca~on exchange resin (Vydac CX 310, The Separation Group, Hesperia, CA) using citrate/acetate buffer pH 5.2, O.12M as eluent and setting the detector potential at + 0.7 V.
TH activity was measured according to the method of Waymire et al. (19), based on the production, trapping and determination of 14CO 2 from carboxyl-/-14C_ _7L-tyrosine and developed by DOPA decarboxylase added to the medium. Enzyme activity was expressed per unit of protein.
Vol. 34, No. 22, 1984
c. /-3H
7-s~i~eroneand
Dopaminergic Function After Chronic L-DOPA
2109
/-3H 7-ADTN bindin$
Z-3H_7-spiperone (New England Nuclear 26.7 Ci/mmol ) binding was determined according to Burt et al. (20). Briefly, rat striata were homogenized by a sonicator in I0 volumes of Tris HCI, 2 mM CaCI^ and i mM MgClp. The crude membrane preparation obtained after centrifuga~ion at 20,000 R g for 20 min w ~ suspended to a final tissue concentration of 3.5 mg wet weight/ml. The b i n d i n g assay was carrie@ out in a total volume of i ~I containing 0.8 ml of buffer (for total binding) or O.i ml (+)butaclamol iO -~ M final concentration (for non specific binding), and O.i ml of 3H-spiperone at concentrations from 0.05 to 2.0 raM. After incubation at 37~C for 15 m i n t h e samples were rapidly filtered under vacuum through Whatman GF/B filters with three 5-ml rinses of cold buffer. 3H-ADTN (New England Nuclear 2 6 . 1 C i / m m l binding was determined according to Creese and Snyder (21). The preparation of crude membranes and the procedure for the binding assay were essentially the same as for 3H-spiperone, with the following modifications: a) the homogenization buffer was only Tris-HCl 50 mM pH 7.7 b) the resuspension buffer included Tris-HCl 50 mM pH 7.1, O.1% ascorbic acid and i mM MnCI 2 c) the incubation time was i0 min at 37°~ All the samples were run in triplicate. Results
In order to see how chronic administration of L-DOPA influenced the normal process of DA release, the change in 3-MT concentration after a single dose of this drug was investigated in rats treated chronically with L-DOPA + benserazide, as described. When L-DOPA + benserazide (IO0 mg/kg + 25 mg/kg p.o.) were given to naive rats, the concentrations of DA and of its methylated metabolite rose by 23% and 110% respectively. A similar increase was observed when the drug was given to L-DOPA + benserazide chronically treated rats (Fig. I). Eff~_~f_~h[~!~_~DOPA_~_~h~_~t~
of TH
Striatal TH activity of normal rats was compared with that of rats chronically treated with L-DOPA. A prolonged increase in DA concentration resulting from chronic L-DOPA did not influenced the basal activity of this enzyme (0.126 + 0.003 pmoles/min/mg protein in controls versus 0.131 + 0.005 in chronically treated animals).
rats To check whether chronic L-DOPA affects the sensitivity of DA receptors, the concentration of 3-MT-normally reduced by treatment with direct DA receptor agonists (12,13,22)°was determined after the administration of apomorphine or piribedil to rats chronically treated with L-DOPA (as described in Methods). Fig. 2 summarizes the effect of injection of 5 mg/kg of apomorphine on striatal 3-MT in controls or chronically L-DOPA treated rats. As shown in the previuos experiment (Fig. I), chronic L-DOPA did not modify the normal concentration of striatal 3-MT. The administration of apomorphine to controls caused a large reduction (66%) in the levels of this DA metabolite; when the drug was given to rats chronically treated with L-DOPA striatal 3-MT was significantly less affected (down 17%) (Fig. 2). Similarly to apomorphine, piribedil (15,30, 60 and
2110
Dopaminergic
ng/g
Function After Chronic L-DOPA
Vol. 34, No. 22, 1984
ng/(
0-6,
3MT
60QO OO
16 000
C
: Controls
Chr
= Chronic
Ac
= Acute
Chr *Ac
_ Chronic - +Acute
o
i
40
14 000
: L
12 00020
T ]7 C
UV- -
Chr
Ac
C
Chr
Chr
AC
~c
hr A
Fig. i Effect of a single dose of L-DOPA on DA and 3-MT in striata of rats chronically treated with L-DOPA + benserazide. L-DOPA + benserazide were administered orally simultaneously as described under Methods. The animals were killed by microwave irradiation 30 minutes after the last L-DOPA + benserazide doses. Data are the mean + S.E. of six determinations. C = controls vehicle + acute L-DOPA + benserazide. Chr + Ac = chronic L-DOPA + benserazide + acute L-DOPA + benserazide.
**
p <0.05 p
versus controls
°°p < O.01 o
versus chronic
p < 0.05
Statistical significance was analyzed by two-way ANOVA test (37) and computer processed by SPBS (23).
(36) and Tukey's
(a)
120 mg/kg, p.o.) in control rats caused a reduction in 3-MT concentration, statistically significant at all the doses tested (Table I) and significantly doserelated (F = -0.55889; p <0.O1). When chronically L-DOPA treated rats were given piribedil, the decrease in 3-MT was dose-related too (F = -0.2856; p (0.05). However, in this case the effect was less pronounced and in fact only at the higher doses (60 and 120 mg/kg) were 3-MT levels significantly reduced (Table I). The slope of the dose/response lines in chronically L-DOPA treated rats (b = -0.0008127 + 0.00051558) and controls(b = 0.002407 + 0.000714) differed significantly, with p
Since the reduction in 3-MT formation may be related to a change in receptor sensitivity, the binding of the antagonist 3H-spiperone and the agonist OH-ADTN to striatal membrane preparations from controls and L-DOPA chronically treated animals was measured. Rats were treated chronically with L-DOPA as described in Methods and binding sites were measured 48 h after the last L-DOPA dose. The specific binding of both 3H-spiperone and 3H-ADTN on striatal membrane prepared from these rats showed a slight but significant decrease (Table 2). This effect was confirmed by three further experiments in which Scatchard analysis
Vol. 34, No. 22, 1984
Dopaminerglc Function After Chronic L-DOPA
ng~g
2111
3MT
30-
C
= Controls
A
= Apomorphine
Chr = C h r o n i c
L-DOPA
C h r ChrOnic L-DOPA + A = +Apornorphine
20-
10-
OO
£
A
ChrChr +A
Fig. 2
Effect of a single dose of apomorphine on 3-MT in striata of controls and rats chronically treated with L-DOPA + benserazide. L-DOPA + benserazide were administered as described in Fig. i. 24 hours after the last dose rats received apomorphine 5 mg/kg and were killed by microwave irradiation 30 minutes thereafter for measurement of striatal 3-MT. Data are the mean + S.E. of six determinations. Statistical significance was analyzed by two-way ANOVA (36) and Tukey's (a) test (37) and computer processed by SPBS (23). p <0.O1
versus apomorphine.
was performed (Fig. 3 gives a representative example). The kinetic constant of these experiments gave an average Bma x value of 0.37 + 0.02 pmole~mg.prot. for L-DOPA treated rats versus a Bma x of 0.44 + 0.01 pmoTes/mg.prot, for the controls (p <0.O5 by Student's t test (24). No--difference was found for the K D values (O.126 + 0.025 nM for the controls and 0.085 + O.O11 nM for the L-DOPA treated group). Discussion The results of these experiments indicate that after chronic treatment with L-DOPA the drug is still able to raise brain concentrations of DA (Fig. i). In fact the concentration of striatal DA after a single L-DOPA injection to rats chronically treated with this drug was not different from that of controis.While this paper was under revision, Melamed et al. (25) reported the results of experiments similar to ours but showing a decrease in DA formation after L-DOPA in rats chronically treated with this drug. We do not know at this moment the reason for this difference. It could depend on the lenght of treatment or it could be due to strain differences. Of course, according to the DA deficit hypothesis of Parkinson's disease, it is pharmacologically important not only that DOPA enhances DA formation but also that the neurotransmitter can be released and interact with the appropriate receptor. We have previously demonstrated that DOPA increases not only DA levels but
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Dopaminergic
Function After Chronic L-DOPA
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also the release of the neurotransmitter as indicated by the rise in 3-MT, (9) the metabolite expressing the amount of DA metabolized in the intersynaptic space (11-13). However, this release related to L - D O P A w a s c l e a r l y different from functional DA release as it was not affected by receptor-mediated feedback mechanisms (9). This finding is confirmed by the present observation that L-DOPA stimulated release of DA was not affected by the prolonged dopaminergic hyperstimulation reciting from chronic treatment with L-DOPA. In fact the 3-MT increase which followed the administration of L-DOPA was equal in control and in chronically L-DOPA treated rats (Fig. i). Table I Effec~ of piribedil on striatal 3-MT of controls and rats chronically treated with L-DOPA Treatment saline
Chronic vehicle
% change
Chronic L-DOPA
% change
11.9+0.7
14.2 + 0.98
piribedil 15 mg/kg
8.3 + i.i ~
- 43%
11.6 + 2.73
-
piribedil 30 mg/kg
5.5 + 0.57
- 61%
12.O + 1.70
+ 0.8%
piribedil 60 mg/kg piribedil 120 mg/kg
6.4 + 0.88 ~
2.5%
- 55%
9.06 + 0.52 ~
-
24%
- 64%
9.8 + 0.64
-
18%
~ 5.2 + 0.32
L-DOPA + benserazide were administered orally simultaneously as described in Methods. 24 h after the last dose rats received piribidil at doses of 15,30, 60 or 120 mg/kg and were killed by microwave irradiation i hour thereafter. Data are the mean + S.E. of six determinations. Statistical signifTcance was analyzed by a modification of Duncan's new multiple test (35). p < 0.O1 versus controls A p < 0.05 versus chronic For statistical
analysis data were transformed as log x.
It has been reported that TH activity can be modified by short and long-term control mechanisms. Short-term regulatory mechanisms involve a change of the enzyme affinity for the substrate or the co-factor (26,27) while the long-term processes, which come into play in cases of intense prolonged sympathetic activity such as stress and reserpine treatment, alter synthesis of the enzyme (28). In the light of these mechanisms we would expect a change in TH after the levels and release of the neurotransmitter had been kept high for a long period. However, the present findings clearly show that these conditions do not modify TH activity. The results discussed so far indicate that chronic hyperstimulation of the dopaminergic system by repeated admnistration of L-DOPA does not affect the presynaptie mechanisms involved in DA synthesis and release.
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Dopamlnerglc Function After Chronic L-DOPA
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Table 2 Specific /-3H. 7-spiperone and /-3H 7-ADTN bindin~ t o striatal membranes of controls and L-DOPA chronically treated rats
31{-spiperone (0.5 l%M) Specific Vehicle
binding
3H-ADTN (4.O nM)
(pmoles/mg
prot.)
.374 + 0.007
.256 + O.012
.329 + 0.008 ~
.214 + 0.012 ~
Chronic L-DOPA
Rats were treated orally twice a day for 24 days with L-DOPA (i00 mg/kg) + benserazide (25 mg/kg) and killed 48 h after the last dose. The values are the mean + S.E. of 14 independent determinations. ~p ~0.05 ~. p ~ 0.01
by Student's t-test.
5 ~r E ..
4
O L. CI
\ \
E
O\
3
\O O
E
Cl
\
2
"W)
~=~
LL
m
\
I
,,* I
I
I
|
° I
I
.050 .100 .150 .200 .2'50 .3'00 .3'50 .400 .450 .500 B (prnoleslmg prot.) Fig. 3 Representative Scatchard plots of 3H-spiperone binding to striatal membranes of controls (@---4) and chronically L-DOPA treated rats (o---o). The mean + S.E. of the binding constant from three individual Scatchard plots were B -- 0.44 + 0.O1 p mol/mg prot and .KD = 0.126 + 0.025 nM for the controls and B maXo.37 +-0.02 p mol/mg/prot and K D = 0.0085--+ 0.011 nM for the L-DOPA treat~Xrats with a statistically significant differenc--e (p~0.05) between the Bmax,analyzed by Student's t-test (24).
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Dopaminergic Function After Chronic L-DOPA
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Feed-back regulation of dopaminergic neurons is known to be under the control of receptor stimulation (5-8), so that their stimulation by an agonist causes inhibition of neuron firing and release of neurotransmitter (5-8,14,29). The opposite is true when DA receptors are blocked or understimulated (6,29). We have previously demonstrated that these feed-back mechanisms on DA release are well reflected by changes in 3-MT (11-13). The fact that the lowering of 3-MT caused by direct DA agonists such as apomorphine and piribedil is attenuated in chronically L-DOPA treated rats indicates that the DA receptors respond less to stimulation (Fig. 2, Table 2). Our in vivo results were partially confirmed. The specific bindinE of both spiperone and ADTN, respectively an antagonist and an agonist of DA receptors, was slightly but significantly reduced. Further experiments with different concentrations of the ligand gave similar results (fig. 4) and calculation of the kinetic constants indicated that the number but not the affinity of the binding sites was affected by chronic L-DOPA. One striking aspect of our results is the fact that while the trend toward down-regulation was evident both in vivo and in vitro, the effect in vivo was much more pronounced (Tab. I and Fig. 2). We feel that the down-regulating effect is exerted specifically on a particular sub-type of DA receptor. As the ligands we used act generally on all known DA receptor subpopulations, an effect on any one of these could be "diluted" by the lack of effect on the others. It is not clear at the moment which receptor sub-type is affected. It has been demonstrated that conformational changes in TH are regulated by post-synaptic receptors (30). Therefore the fact that TH activity was not changed in L-DOPA treated rats seems to exclude the possibility that these are the receptors that are down regulated. However, further experimental support is needed to verify this. Our findings indicate tha~ although in our conditions, chronic administration of L-DOPA did not modify the increase in DA formation and release after a single injection of the drug, it did indeed cause a change in the functional properties of dopaminergic nerve terminals. This down-regulation after chronic L-DOPA confirms previous observations by Mishra and co-workers (29), by List and Seeman (31) and by Cimino et al. (8) but is in contrast with the work of Suga (32), who found no change in receptor binding after chronic L-DOPA, and other authors who even reported supersensitivity (33-34). It is difficult to explain these conflicting reports. Differences in the doses used or'routes of administration, such as mixing L-DOPA with diet (33), may be at the basis of the different effect. Recently for instance Wilner et al. (34) showed an increase of 3H-spiperone binding to membranes after repeated high doses of L-DOPA mixed with the diet and interpreted this as a possible blocking action On the DA receptor by a tetraisoquinoline, compound formed from exogenous L~DOPA. We recently tested the effect on 3H-spiperone binding sites of chronic doses of L-DOPA ranging from 20 to i00 mg/kg and always found a decrease of the binding sites (unpublished data). Up to now our results indicate that L-DOPA given repeatedly at doses that maximally raise the concentration and release of brain DA causes a down-regulation of DA receptors. The possibility that these phenomena may underly some of the drawbacks of L-DOPA therapy, especially the progressive loss of efficacy, requires further serious consideration. Acknowledgements This work was supported by a grant from the National Research Council no. CT 81.O0258.O4. We thank Drs. Da Prada and Merzario of Hoffmann La Roche Inc. fo~ the generous supply of benserazide.
Vol. 34, No. 22, 1984
Dopaminergic Function After Chronic L-DOPA
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References
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32. M. SUGA, Life Sci. 27 877-882 (1980). 33. C. PYCOCK, D. DAWBARN and C. O'SHAUGHNESSY, Eur. J. Pharmac. 79 201-215 (1982). 34. K.D. WILNER, I.J. BUTLER, W.E. SEIFERT Jr. and Y.C. CLEMENT-CORMIER, Biochem. Pharmac. 29 701-706 (1980). 35. C.Y. KRAMER, Biometrics 12 307-310 (1956). 36. R.E. KIRK, Experimental Design Procedures for the Behavioral Sciences, Brooks, Belmont, California (1968). 37. D.V. CICCHETTI, Psychol. Bull. 77 405-408 (1972).
Pergamon Press
IN VIVO AND IN VITRO EVIDENCE OF DOPAMINERGIC SYSTEM DOWN REGULATION INDUCED BY CHRONIC L-DOPA
F. Ponzio, M. Cimino, G. Achilli, M. Lipartiti, C. Perego, G. Vantini and S. Alger~ Istituto di Ricerche Farmacologiche '~ario Negri" - via Eritrea, 62 20157 Milan, Italy. (Received in final form March 19, 1984) Summary The biochemical modifications which occur in the dopaminergic system after chronic administration of L-DOPA are investiga~e~ Levels of DA and of its metabolite 3-methoxytyramine (3-MT), an expression of the amount of DA released, were raised to the same extent in controls given a single dose of I-DOPA and in chronically treated rats given iOO mg/kg of I-DOPA plus 25 mg/kg of benserazide twice a day for 24 days. However, the reduction in neuronal function expressed by the decrease in 3-MT which follows treatment with DA agonists such as piribedil and apomorphine was less pronounced in the chronically L-DOPA treated rats. This suggests that such treatment causes a down regulation of DA receptors. These in vivo results were confirmed by in vitro analysis of DA receptor activity after chronic L-DOPA. Under these conditions there was a significant reduction in the number of L-3H_7-spiperone and L-3H_/-ADTN binding sites with no changes in their affinity. The in vivo and in vitro findings both suggest the involvement of a subsensitive compensatory mechanism or down regulation of dopaminergic neurons after chronic treatment with L-DOPA. Long-term levodopa (L-DOPA) therapy in Parkinson's disease is often associated with a decrease in the efficacy of this drug (1-4). The causes of this"tolerance" remain largely unclarified although on a theoretical basis the hypothesis can be formulated of a down-regulatory of some of the mechanisms of dopaminergic transmission, brought about by chronic dopaminergic hyperstimulation (5-8). In fact L-DOPA raises the concentration of dopamine (DA) at receptor sites (9) and therefore it might be considered an indirect dopaminergic agonist (iO). Experiments to date to test the possibility that chronic L-DOPA reduces the sensitivity of dopaminergic receptors have given conflicting results. Two levels of action should be considemed in tackling this question. One is at the presynapse: chronic hyperfunction could result in less release of DA formed from exogenous L-DOPA or it could affect tyrosine hydroxylase so that the activity of this enzyme remains inhibited beyond the period during which the concentration of DA is high. The other possible level of action of chronic L-DOPAis at the receptors: chronic hyperstimulation of DA receptors could in fact render them hyposensitive, thus explaining the decreased response to the neurotransmitter even when a high concentration is present. iCorrespondence
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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Dopaminergic Function After Chronic L-DOPA
Vol. 34, No. 22, 1984
We have tested the two hypothesis in rats treated chronically with L-DOPA by determining i) whether the formation and release of DA after a single dose of L-DOPA and after chronic treatment is the same or not - DA release was evaluated by measuring changes in the concentration of 3-methoxy-4-hydroxytyramine (3-MT) ~, an intrasynaptic DA metabolite that reflects the amount of DA present in the synaptic cleft (9,11-13); 2) whether chronic treatment influences the activity of tyrosine hydroxylase (TH); 3) whether there is any change in DA receptor sensitivity. Our investigations included changes in specific binding of DA agonists and antagonists, and the efficiency of the negative feed-back response which usually follows stimulation of dopaminergic receptors by agonist drugs (9,14,15). Materials and Methods Animals CD-COBS male rats (Charles River, Italy) weighing 175 + 20 g were housed under standard conditions with free access to food and water. They were treated orally twice a day for 24 days with a suspension in 0.5% carboxymethylcellulose (C.M.C.) of i00 mg/kg or L-DOPA and 25 mg/kg of benserazide, an inhibitor of peripheral decarboxylase (16). Chronically L-DOPA + benserazide treated rats were given the following drugs as single doses, pyribedil, apomorphine and pargyline. Rats were killed 24 hours after last L-DOPA dose for determination of the activity of the enzyme TH and of DA and its metabolites, or 48 hafter the last L-DOPA dose for determining /-3H 7-spiperone or ~-3H_7-ADTN receptor binding. Rats were killed by d$cap~tation except when DA and its metabolites had to be assayed, in which case microwave irradiation (1.3 at 2.45 GHz for 4.25 sec) was used in order to rapidly inactivate catechol-o-methyl-transferase (COMT; E.C. 2.11.6) (17). In boyh cases striata were rapidly dissected, frozen on dry ice and kept at -80°C until biochemical assay.
a. DA and 3-MT For the simultaneous assay of 3-MT and DA the striatum of one animal was homogenized by sonifier in 300 ~i of 0.4N perchloric acid and centrifuged. The clear supernatant was adjusted to pH 7.5 - 7.6 to separate DA from 3-MT. At this pH DA was adsorbed onto alumina and 3-MT remained in the supernatant from which it was extracted according to our previously described methods (11,12). DA was eluted from the alumina according to Keller et al. (18). Liquid chromatography with electrochemical detection (LCEC) (Bioanalytical System Inc., West Lafayette, Ind.) was used for all biochemical assays. A glass column 500 n~n long, 2 mm i.d., packed with a cation exchange resin (Vydac CX, P310), The Separation Group Experia, CA) was used for DA determination. Samples were eluted with citrate/acetate buffer pH 5.2, 0.03 M; the detector was set at a potential of + 0.75 V. 3-MT was separated in a glass column 300 mm long, 2 mm i.d., packed with ca~on exchange resin (Vydac CX 310, The Separation Group, Hesperia, CA) using citrate/acetate buffer pH 5.2, O.12M as eluent and setting the detector potential at + 0.7 V.
TH activity was measured according to the method of Waymire et al. (19), based on the production, trapping and determination of 14CO 2 from carboxyl-/-14C_ _7L-tyrosine and developed by DOPA decarboxylase added to the medium. Enzyme activity was expressed per unit of protein.
Vol. 34, No. 22, 1984
c. /-3H
7-s~i~eroneand
Dopaminergic Function After Chronic L-DOPA
2109
/-3H 7-ADTN bindin$
Z-3H_7-spiperone (New England Nuclear 26.7 Ci/mmol ) binding was determined according to Burt et al. (20). Briefly, rat striata were homogenized by a sonicator in I0 volumes of Tris HCI, 2 mM CaCI^ and i mM MgClp. The crude membrane preparation obtained after centrifuga~ion at 20,000 R g for 20 min w ~ suspended to a final tissue concentration of 3.5 mg wet weight/ml. The b i n d i n g assay was carrie@ out in a total volume of i ~I containing 0.8 ml of buffer (for total binding) or O.i ml (+)butaclamol iO -~ M final concentration (for non specific binding), and O.i ml of 3H-spiperone at concentrations from 0.05 to 2.0 raM. After incubation at 37~C for 15 m i n t h e samples were rapidly filtered under vacuum through Whatman GF/B filters with three 5-ml rinses of cold buffer. 3H-ADTN (New England Nuclear 2 6 . 1 C i / m m l binding was determined according to Creese and Snyder (21). The preparation of crude membranes and the procedure for the binding assay were essentially the same as for 3H-spiperone, with the following modifications: a) the homogenization buffer was only Tris-HCl 50 mM pH 7.7 b) the resuspension buffer included Tris-HCl 50 mM pH 7.1, O.1% ascorbic acid and i mM MnCI 2 c) the incubation time was i0 min at 37°~ All the samples were run in triplicate. Results
In order to see how chronic administration of L-DOPA influenced the normal process of DA release, the change in 3-MT concentration after a single dose of this drug was investigated in rats treated chronically with L-DOPA + benserazide, as described. When L-DOPA + benserazide (IO0 mg/kg + 25 mg/kg p.o.) were given to naive rats, the concentrations of DA and of its methylated metabolite rose by 23% and 110% respectively. A similar increase was observed when the drug was given to L-DOPA + benserazide chronically treated rats (Fig. I). Eff~_~f_~h[~!~_~DOPA_~_~h~_~t~
of TH
Striatal TH activity of normal rats was compared with that of rats chronically treated with L-DOPA. A prolonged increase in DA concentration resulting from chronic L-DOPA did not influenced the basal activity of this enzyme (0.126 + 0.003 pmoles/min/mg protein in controls versus 0.131 + 0.005 in chronically treated animals).
rats To check whether chronic L-DOPA affects the sensitivity of DA receptors, the concentration of 3-MT-normally reduced by treatment with direct DA receptor agonists (12,13,22)°was determined after the administration of apomorphine or piribedil to rats chronically treated with L-DOPA (as described in Methods). Fig. 2 summarizes the effect of injection of 5 mg/kg of apomorphine on striatal 3-MT in controls or chronically L-DOPA treated rats. As shown in the previuos experiment (Fig. I), chronic L-DOPA did not modify the normal concentration of striatal 3-MT. The administration of apomorphine to controls caused a large reduction (66%) in the levels of this DA metabolite; when the drug was given to rats chronically treated with L-DOPA striatal 3-MT was significantly less affected (down 17%) (Fig. 2). Similarly to apomorphine, piribedil (15,30, 60 and
2110
Dopaminergic
ng/g
Function After Chronic L-DOPA
Vol. 34, No. 22, 1984
ng/(
0-6,
3MT
60QO OO
16 000
C
: Controls
Chr
= Chronic
Ac
= Acute
Chr *Ac
_ Chronic - +Acute
o
i
40
14 000
: L
12 00020
T ]7 C
UV- -
Chr
Ac
C
Chr
Chr
AC
~c
hr A
Fig. i Effect of a single dose of L-DOPA on DA and 3-MT in striata of rats chronically treated with L-DOPA + benserazide. L-DOPA + benserazide were administered orally simultaneously as described under Methods. The animals were killed by microwave irradiation 30 minutes after the last L-DOPA + benserazide doses. Data are the mean + S.E. of six determinations. C = controls vehicle + acute L-DOPA + benserazide. Chr + Ac = chronic L-DOPA + benserazide + acute L-DOPA + benserazide.
**
p <0.05 p
versus controls
°°p < O.01 o
versus chronic
p < 0.05
Statistical significance was analyzed by two-way ANOVA test (37) and computer processed by SPBS (23).
(36) and Tukey's
(a)
120 mg/kg, p.o.) in control rats caused a reduction in 3-MT concentration, statistically significant at all the doses tested (Table I) and significantly doserelated (F = -0.55889; p <0.O1). When chronically L-DOPA treated rats were given piribedil, the decrease in 3-MT was dose-related too (F = -0.2856; p (0.05). However, in this case the effect was less pronounced and in fact only at the higher doses (60 and 120 mg/kg) were 3-MT levels significantly reduced (Table I). The slope of the dose/response lines in chronically L-DOPA treated rats (b = -0.0008127 + 0.00051558) and controls(b = 0.002407 + 0.000714) differed significantly, with p
Since the reduction in 3-MT formation may be related to a change in receptor sensitivity, the binding of the antagonist 3H-spiperone and the agonist OH-ADTN to striatal membrane preparations from controls and L-DOPA chronically treated animals was measured. Rats were treated chronically with L-DOPA as described in Methods and binding sites were measured 48 h after the last L-DOPA dose. The specific binding of both 3H-spiperone and 3H-ADTN on striatal membrane prepared from these rats showed a slight but significant decrease (Table 2). This effect was confirmed by three further experiments in which Scatchard analysis
Vol. 34, No. 22, 1984
Dopaminerglc Function After Chronic L-DOPA
ng~g
2111
3MT
30-
C
= Controls
A
= Apomorphine
Chr = C h r o n i c
L-DOPA
C h r ChrOnic L-DOPA + A = +Apornorphine
20-
10-
OO
£
A
ChrChr +A
Fig. 2
Effect of a single dose of apomorphine on 3-MT in striata of controls and rats chronically treated with L-DOPA + benserazide. L-DOPA + benserazide were administered as described in Fig. i. 24 hours after the last dose rats received apomorphine 5 mg/kg and were killed by microwave irradiation 30 minutes thereafter for measurement of striatal 3-MT. Data are the mean + S.E. of six determinations. Statistical significance was analyzed by two-way ANOVA (36) and Tukey's (a) test (37) and computer processed by SPBS (23). p <0.O1
versus apomorphine.
was performed (Fig. 3 gives a representative example). The kinetic constant of these experiments gave an average Bma x value of 0.37 + 0.02 pmole~mg.prot. for L-DOPA treated rats versus a Bma x of 0.44 + 0.01 pmoTes/mg.prot, for the controls (p <0.O5 by Student's t test (24). No--difference was found for the K D values (O.126 + 0.025 nM for the controls and 0.085 + O.O11 nM for the L-DOPA treated group). Discussion The results of these experiments indicate that after chronic treatment with L-DOPA the drug is still able to raise brain concentrations of DA (Fig. i). In fact the concentration of striatal DA after a single L-DOPA injection to rats chronically treated with this drug was not different from that of controis.While this paper was under revision, Melamed et al. (25) reported the results of experiments similar to ours but showing a decrease in DA formation after L-DOPA in rats chronically treated with this drug. We do not know at this moment the reason for this difference. It could depend on the lenght of treatment or it could be due to strain differences. Of course, according to the DA deficit hypothesis of Parkinson's disease, it is pharmacologically important not only that DOPA enhances DA formation but also that the neurotransmitter can be released and interact with the appropriate receptor. We have previously demonstrated that DOPA increases not only DA levels but
2112
Dopaminergic
Function After Chronic L-DOPA
Vol. 34, No. 22, 1984
also the release of the neurotransmitter as indicated by the rise in 3-MT, (9) the metabolite expressing the amount of DA metabolized in the intersynaptic space (11-13). However, this release related to L - D O P A w a s c l e a r l y different from functional DA release as it was not affected by receptor-mediated feedback mechanisms (9). This finding is confirmed by the present observation that L-DOPA stimulated release of DA was not affected by the prolonged dopaminergic hyperstimulation reciting from chronic treatment with L-DOPA. In fact the 3-MT increase which followed the administration of L-DOPA was equal in control and in chronically L-DOPA treated rats (Fig. i). Table I Effec~ of piribedil on striatal 3-MT of controls and rats chronically treated with L-DOPA Treatment saline
Chronic vehicle
% change
Chronic L-DOPA
% change
11.9+0.7
14.2 + 0.98
piribedil 15 mg/kg
8.3 + i.i ~
- 43%
11.6 + 2.73
-
piribedil 30 mg/kg
5.5 + 0.57
- 61%
12.O + 1.70
+ 0.8%
piribedil 60 mg/kg piribedil 120 mg/kg
6.4 + 0.88 ~
2.5%
- 55%
9.06 + 0.52 ~
-
24%
- 64%
9.8 + 0.64
-
18%
~ 5.2 + 0.32
L-DOPA + benserazide were administered orally simultaneously as described in Methods. 24 h after the last dose rats received piribidil at doses of 15,30, 60 or 120 mg/kg and were killed by microwave irradiation i hour thereafter. Data are the mean + S.E. of six determinations. Statistical signifTcance was analyzed by a modification of Duncan's new multiple test (35). p < 0.O1 versus controls A p < 0.05 versus chronic For statistical
analysis data were transformed as log x.
It has been reported that TH activity can be modified by short and long-term control mechanisms. Short-term regulatory mechanisms involve a change of the enzyme affinity for the substrate or the co-factor (26,27) while the long-term processes, which come into play in cases of intense prolonged sympathetic activity such as stress and reserpine treatment, alter synthesis of the enzyme (28). In the light of these mechanisms we would expect a change in TH after the levels and release of the neurotransmitter had been kept high for a long period. However, the present findings clearly show that these conditions do not modify TH activity. The results discussed so far indicate that chronic hyperstimulation of the dopaminergic system by repeated admnistration of L-DOPA does not affect the presynaptie mechanisms involved in DA synthesis and release.
Vol. 34, No. 22, 1984
Dopamlnerglc Function After Chronic L-DOPA
2113
Table 2 Specific /-3H. 7-spiperone and /-3H 7-ADTN bindin~ t o striatal membranes of controls and L-DOPA chronically treated rats
31{-spiperone (0.5 l%M) Specific Vehicle
binding
3H-ADTN (4.O nM)
(pmoles/mg
prot.)
.374 + 0.007
.256 + O.012
.329 + 0.008 ~
.214 + 0.012 ~
Chronic L-DOPA
Rats were treated orally twice a day for 24 days with L-DOPA (i00 mg/kg) + benserazide (25 mg/kg) and killed 48 h after the last dose. The values are the mean + S.E. of 14 independent determinations. ~p ~0.05 ~. p ~ 0.01
by Student's t-test.
5 ~r E ..
4
O L. CI
\ \
E
O\
3
\O O
E
Cl
\
2
"W)
~=~
LL
m
\
I
,,* I
I
I
|
° I
I
.050 .100 .150 .200 .2'50 .3'00 .3'50 .400 .450 .500 B (prnoleslmg prot.) Fig. 3 Representative Scatchard plots of 3H-spiperone binding to striatal membranes of controls (@---4) and chronically L-DOPA treated rats (o---o). The mean + S.E. of the binding constant from three individual Scatchard plots were B -- 0.44 + 0.O1 p mol/mg prot and .KD = 0.126 + 0.025 nM for the controls and B maXo.37 +-0.02 p mol/mg/prot and K D = 0.0085--+ 0.011 nM for the L-DOPA treat~Xrats with a statistically significant differenc--e (p~0.05) between the Bmax,analyzed by Student's t-test (24).
2114
Dopaminergic Function After Chronic L-DOPA
Vol. 34, No. 22, 1984
Feed-back regulation of dopaminergic neurons is known to be under the control of receptor stimulation (5-8), so that their stimulation by an agonist causes inhibition of neuron firing and release of neurotransmitter (5-8,14,29). The opposite is true when DA receptors are blocked or understimulated (6,29). We have previously demonstrated that these feed-back mechanisms on DA release are well reflected by changes in 3-MT (11-13). The fact that the lowering of 3-MT caused by direct DA agonists such as apomorphine and piribedil is attenuated in chronically L-DOPA treated rats indicates that the DA receptors respond less to stimulation (Fig. 2, Table 2). Our in vivo results were partially confirmed. The specific bindinE of both spiperone and ADTN, respectively an antagonist and an agonist of DA receptors, was slightly but significantly reduced. Further experiments with different concentrations of the ligand gave similar results (fig. 4) and calculation of the kinetic constants indicated that the number but not the affinity of the binding sites was affected by chronic L-DOPA. One striking aspect of our results is the fact that while the trend toward down-regulation was evident both in vivo and in vitro, the effect in vivo was much more pronounced (Tab. I and Fig. 2). We feel that the down-regulating effect is exerted specifically on a particular sub-type of DA receptor. As the ligands we used act generally on all known DA receptor subpopulations, an effect on any one of these could be "diluted" by the lack of effect on the others. It is not clear at the moment which receptor sub-type is affected. It has been demonstrated that conformational changes in TH are regulated by post-synaptic receptors (30). Therefore the fact that TH activity was not changed in L-DOPA treated rats seems to exclude the possibility that these are the receptors that are down regulated. However, further experimental support is needed to verify this. Our findings indicate tha~ although in our conditions, chronic administration of L-DOPA did not modify the increase in DA formation and release after a single injection of the drug, it did indeed cause a change in the functional properties of dopaminergic nerve terminals. This down-regulation after chronic L-DOPA confirms previous observations by Mishra and co-workers (29), by List and Seeman (31) and by Cimino et al. (8) but is in contrast with the work of Suga (32), who found no change in receptor binding after chronic L-DOPA, and other authors who even reported supersensitivity (33-34). It is difficult to explain these conflicting reports. Differences in the doses used or'routes of administration, such as mixing L-DOPA with diet (33), may be at the basis of the different effect. Recently for instance Wilner et al. (34) showed an increase of 3H-spiperone binding to membranes after repeated high doses of L-DOPA mixed with the diet and interpreted this as a possible blocking action On the DA receptor by a tetraisoquinoline, compound formed from exogenous L~DOPA. We recently tested the effect on 3H-spiperone binding sites of chronic doses of L-DOPA ranging from 20 to i00 mg/kg and always found a decrease of the binding sites (unpublished data). Up to now our results indicate that L-DOPA given repeatedly at doses that maximally raise the concentration and release of brain DA causes a down-regulation of DA receptors. The possibility that these phenomena may underly some of the drawbacks of L-DOPA therapy, especially the progressive loss of efficacy, requires further serious consideration. Acknowledgements This work was supported by a grant from the National Research Council no. CT 81.O0258.O4. We thank Drs. Da Prada and Merzario of Hoffmann La Roche Inc. fo~ the generous supply of benserazide.
Vol. 34, No. 22, 1984
Dopaminergic Function After Chronic L-DOPA
2115
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Dopaminergic Function After Chronic L-DOPA
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