Proc. Nati. Acad. Sci. USA Vol. 75, No. 6, pp. 2641-2643, June 1978

Biochemistry

Conformation of dopamine at the dopamine receptor (conformationally restricted analogs/dopamine receptor binding/corpus striatum)

HAROLD L. KOMISKEY, JOSEF F. BOSSART, DUANE D. MILLER, AND POPAT N. PATIL* Divisions of Pharmacology and Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210

Communicated by Melvin S. Newnan, March 15,1978

ABSTRACT Tritiated dopamine was used to label the dopamine receptor in membranes isolated from the rat corpus striatum. Scatchard analysis of displacement of [3Hldopamine by nonradioactive dopamine indicated the presence of two binding sites. The similarities in affinity, capacity, and drug specificity of the high-affinity site in the striatal membranes from rat and the binding site in the membranes from the calf caudate nucleus suggest that [3Hjdopamine labels the same site in both species. In order to determine what conformation of dopamine is preferred at the dopamine receptor site, conformationally restricted analogs of dopamine-namely, the cis and trans 2-amino-1(3,4-dihydroxyphenyl)cyclobutane hydrochlorides-were tested for their affinity to the receptor. Compared to the cis conformation, the trans-restricted analogs had more affinity for the receptor site, indicating that dopamine probably interacts with the receptor in the trans conformation.

There is considerable interest in the relationship between conformation of neurotransmitters at their receptors and biological activity (1, 2). The putative neurotransmitter dopamine is probably involved in parkinsonism (3), Huntington chorea (4), drug-induced dyskinesia (5, 6), schizophrenia (7), and manic depressive psychosis (8, 9). The preferred conformation of dopamine in the solid state is the trans extended form, and in solution it exists primarily in the trans and gauche conformations with the trans form being slightly predominant (10, 11). However, it is realized that the preferred conformation of dopamine in the dopamine-receptor complex need not be the same as that in solution or in the solid state (11, 2). Nevertheless, investigations with dopamine-sensitive adenylate cyclase support the idea that the trans extended form of dopamine interacts with the dopamine receptor. Among the semirigid analogs of dopamine, the compounds with the greatest potency are those with the ethylamine side chain of dopamine in the fully extended form, as in apomorphine and 2-amino-6,7dihydroxytetralin (12-16). On the other hand, there are significant differences indrugaffinity, subcellular localization, and ontogenetic developmental pattern between the dopamine receptor recognition site and adenylate cyclase (17-20). Thus, the significance of the dopamine-sensitive adenylate cyclase data in relation to dopamine-mediated behaviors is not known. Apomorphine and 2-amino-6,7-dihydroxytetralin have an affinity for the dopamine receptor binding site as great or greater than that of dopamine (21). This affinity, however, may not be due to these compounds having the dopamine moiety in a fully extended form, because no binding study has been carried out comparing the affinity of isomeric compounds that possess a cis and trans relationship between the catechol and amino groups found in dopamine. Therefore, to determine the preferred conformation of dopamine at its receptor site, we The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

determined the affinity of conformationally restricted trans and cis analogs of dopamine at [3H]dopamine binding sites. MATERIALS AND METHODS of Preparation the homogenate and the dopamine binding assay was performed similar to the method of Burt et al. (21). The corpora striata of four Sprague-Dawley rats (200-300 g) were homogenized in 40 vol of cold 50 mM Tris buffer (pH 7.7) at 250C, with a Brinkman Polytron PT-10 (setting 6,5 sec, twice). The homogenate was centrifuged twice at 50,000 X g for 10 min with rehomogenization (setting 6, 5 sec) of the intermediate pellet in fresh buffer. The final pellet was homogenized in 90 vol of cold 50 mM Tris/0. 1% ascorbic acid/10,uM iproniazid/ 120 mM NaCl/5 mM KC1/2 mM CaCi2/1 mM MgCi2, to give a final pH of 7.1 at 37°C. This homogenate was placed in a 370C bath for 5 min and then returned to ice. Incubations (10 min, 370C) were carried out in a 2-ml final volume containing 10 nM [3H]dopamine (16.409 or 21.421 Ci/mmol, New England Nuclear) which was added in 100 gl of 0.1% ascorbic acid, 1.8 ml of tissue suspension, and 100 ,1 of various concentrations of drugs dissolved in 0.1% ascorbic acid. Incubations were terminated by filtration under vacuum through Whatman GF/B filters with two 5-ml rinses of cold buffer. The filters were placed in 20-ml scintillation vials containing 10 ml of Thrift Solve liquid scintillation cocktail (Kew Scientific). The vials were mechanically shaken for 60 min to remove the [3H]dopamine from the filters and thereby minimize self-absorption of tritium. The radioactivity of each sample was assayed in a Beckman liquid scintillation spectrometer, model LS-345. Quench was monitored by use of an automatic external standard. Counting efficiencies were between 35 and 40%. All the drugs in higher concentrations decreased binding of [3H]dopamine to the glass fiber filters, so an equal number of samples were prepared identical to those described above except that the membranes were omitted. The [3H]dopamine retained by the filters under these conditions was subtracted from the corresponding sample containing the membranes. The dose-dependent decreases in [3H]dopamine binding were analyzed by linear regression (22, 23) after transformation of the data to probits (24). Nonradioactive dopamine was purchased from Regis Chemical Co., Chicago, IL. Iproniazid phosphate was purchased from Hoffmann-LaRoche, Inc., Nutley, NJ. The trans and cis isomers of 2-amino-1-(3,4-dihydroxyphenyl)cyclobutane hydrobromide and NN-dimethyl-2-amino-1-(3,4-dihydroxyphenyl)cyclobutane hydrobromide were synthesized in our medicinal chemistry laboratory. RESULTS Increasing concentrations of nonradioactive dopamine (1 nM-100 AM), [3H]dopamine, and striatal membranes were used *

2641

To whom reprint requests should be addressed.

Biochemistry: Komiskey et al.

2642 1.0

Proc. Natl. Acad. Sci. USA 75 (1978) Table 1. Affinity of conformationally restricted analogs of dopamine for the dopamine receptor Ki (mean 41 SEM),

A

0.9

E 0.8 . 'a 0.7, E 0.6

HO

0.. nZ

n

0.0256 i 0.005

7

OH

0.4

o

0 I-

, 0.3 , S 0.2

AM

I

NH2 DA

0.1

I.:

HO

.. -..

300

100

200

500

700

OH

900

1100 1300 400 600 800 1000 1200 1400 Dopamine bound, pmol/g

oi

H

8.16

1.71

6

f"

16

H

B

x

Ea 14

HO

OH

i,c 12 .s

NH,

trats- I

NH,

160.19 4 50.11

6

0.529 i 0.056

7

18.34 + 2.19

6

10 _

.0

*c

8

H

_

a

6

cis-I H~w OH

I

4

o

E

-; 2

H

H

(CH,)2

u

a

co)

10

20

30

40

[3H]Dopamine,

50

60

trans-If HO OH

nM

FIG. 1. (A) Scatchard plot obtained by adding increasing concentrations of nonradioactive dopamine to vials containing 10 nM [3H]dopamine and rat corpus striatum membranes. K1 (2.17 X 10-8 M) and K2 (5.64 X 10-6 M) are the dissociation constants for the higher and lower affinity sites, respectively, with maximum binding capacities, Bmax, and Bmas2, of 22.97 and 1266.57 pmol/g, respectively. Points are means of seven observations. (B) Saturation of specific [3H]dopamine binding. Increasing concentrations of [3H]dopamine were incubated with membranes of rat corpus striatum for 10 min. Points are the means (±SEM) of five observations. to obtain the Scatchard plot. The Scatchard plot was a curve that resolved into two linear components according to the methods of Klotz and Hunston (25) and Vallner et al. (26). The lower affinity site has a dissociation constant of 5.64 AM. The higher affinity site has a dissociation constant of 21.69 nM, with

oC

N(CH3)2

cis-II

The concentrations of drugs required to inhibit specific binding of dopamine (DA) by 50% (IC50) were determined by log-probit analysis and converted to Ki values by the formula Ki = IC50/(1 + c/KD), in which c is the concentration of [3H]dopamine (10 nM) and Kd is its dissociation constant (21.69 nM). Specific [3H]dopamine binding was defined as that portion of total binding displaced by 1 MAM unlabeled dopamine.

was

approximately 1/6o the number of binding sites as the lower affinity site (Fig. 1A). To limit binding mainly to the higher 7 DA

trans- I

i

6

trans-I

95

cis-II cis-I

c

3

i

0

80 o 60 50 E 0

5

C

0

20

4'

a

I

o0

0

5 3

0)OL

._

Ils1 III

1 10-9

i

o8

[III[

IIII

IIII

i-5 Concentration, M

1o

10-6

IlIII

o-o4

1111l _j l-3

W

FIG. 2. Dose-dependent decrease in [3H]dopamine binding caused by restricted analogs of dopamine (DA). Lines were drawn by linear regression after transformation of the data to probits. Points are the means of six or seven observations. See text for analogs.

affinity site, the amount of radioactivity displaced from the total bound by 1 ,gM dopamine (approximately 35%) was used and termed "specific" binding. The [3H]dopamine binding is saturable (Fig. 1B). The conformationally restricted analogs of dopamine produced parallel shifts in decreasing [3H]dopamine binding with respect to the dose-dependent decrease elicited by nonradioactive dopamine (Fig. 2). However, cis-N,N-dimethyl-2amino-1-(3,4-dihydroxyphenyl)cyclobutane (cis-II) at higher concentrations produced a nonparallel dose-dependent decrease in [3H]dopamine binding. Trans-2-amino-1-(3,4-dihydroxyphenyl)cyclobutane (trans-I) had about 20 times more affinity for the [3H]dopamine binding site than did the cis-I isomer (Table 1). The addition of an N-dimethyl group to the above geometric isomers increased the affinity of the molecules, especially the trans isomer, for the [3H]dopamine binding site. The affinity of trans-N,N-dimethyl-2-amino-1-(3,4-dihydroxyphenyl)cyclobutane (trans-II) was approximately 35 times that of cis isomer (cis-II). When examined on the synaptosomes isolated from either rat brain cortex or striatum, these substances also inhibited the uptake of catecholamines, but the concentration required for the inhibition was high. Again, the trans form was a more ef-

Biochemistry: Komiskey et al.

Proc. Nati. Acad. Sci. USA 75 (1978)

B HO

cules caused by N-substitution illustrates the possibility of obtaining more potent and specific dopamine agonists. This study was supported in part by U.S. Public Health Service Grants GM-17859 and NS 09350 from the National Institutes of Health, Bethesda, MD.

A H

O

*

0 1 C

HQ H

2643

SH

z

110-,

1600 HO H

Q

1. Patil, P. N., Miller, D. D. & Trendelenburg, U. (1974) Pharmacol. Rev. 26, 323-392. 2. Maxwell, R. A., Ferris, R. M. & Burcsu, J. E. (1976) in The Mechanism of Neuronal and Extraneuronal Transport of Catecholamines, ed. Paton, D. M. (Raven, New York), pp.

95-153.

O Lso ISO

wv--.. -30*, 0 IZ IO" Newman Trans-extended FIG. 3. projections. (A) conformation of dopamine. The outer limits for the dihedral angles are illustrated for both trans-I (B) and cis-I (C). -

fective inhibitor than the cis form (27). The conformational requirements of drugs for the "transport" receptor and the postsynaptic receptor appear to be similar.

DISCUSSION The affinity, total number of binding sites, and drug specificity at the high-affinity site in membranes from rat corpus striatum are similar to the values reported for the calf caudate nucleus (21). Therefore, under the conditions of these experiments, [3H]dopamine probably labels the same site in both species, presumably the postsynaptic receptor site for dopamine. The parallel shifts in the decrease in [3H]dopamine binding caused by several restricted analogs of dopamine with respect to the dose-dependent decrease elicited by nonradioactive dopamine indicates that the compounds are binding to the same site (Fig. 2). Cis-II has the ability to bind to an additional binding site or to bind to the receptor in'a different manner, because at higher concentrations it produced a nonparallel dose-dependent decrease in [3H]dopamine binding. The major finding of this study is the greater affinity of the trans restricted analogs of dopamine for the [3H]dopamine binding site (Table 1). These results strongly suggest that dopamine interacts with its receptor in the trans conformation. It should be noted that the compounds possessing a trans configuration (trans-I and trans-II) are conformationally restricted and the dihedral angle between the important functional groups (namely, the amino and catechol groups) can exist within a range of -1 10°-1600; the compounds possessing a cis configuration (cis-I and cis-II) possess dihedral angles in the range of 100 to -300 as illustrated in Fig. 3. Thus, the compound possessing the highest affinity for the binding site in both the primary and tertiary analogs more closely approximates the trans-extended form of dopamine (dihedral angle, 180°) which has been proposed to be the form of dopamine that interacts best with dopamine receptors (28). Previously, the addition of N-dialkyl groups to restricted analogs of dopamine has been shown to increase their potency on behavioral test for dopamine agonist activity (29-32). Therefore, it was not surprising that the addition of N-dimethyl groups to the geometric isomers of 2-amino-1-(3,4-dihydroxyphenyl)cyclobutane increased their affinity for the [3H]dopamine binding site. This increase in affinity of the rigid mole-

3. Hornykiewicz, 0. (1974) Life Sci. 15, 1249-1260. 4. Klawans, H. L. (1973) The Pharmacology of Extrapyramidal Movement Disorders (Karger, Basel), pp. 71-83. 5. Villineuve, A. (1972) Can. Psychlatr. Assoc. J. 17 (Suppl.), 69-72. 6. Klawans, H. L. (1973) Am. J. Psychiatry 130,82-86. 7. Hornykiewicz, 0. (1977) Ann. Rev. Pharmacol. Toxicol. 17, 545-559. 8. Bunney, W. E. & Murphy, D. L. (1974) in Factors in Depression, ed. Kline, N. S. (Raven, New York), pp. 139-158. 9. Murphy, D. L. & Redmond, D. E. (1975) in Catecholamines and Behavior, ed. Friedhoff, A. J. (Plenum, New York), Vol. 2, pp. 73-117. 10. Bergin, R. & Carlstrom, D. (1968) Acta Crystallogr. B-24, 1506-1510. 11. Bustard, T. M. & Egan, R. S. (1971) Tetrahedron 27, 44574469. 12. Recker, R. F., Engel, D. J. C. & Nys, G. G. (1972) J. Pharm.

Pharmacol. 24,589-591. 13. Miller, R. J., Horn, A. S., Iversen, L. L. & Pinder, R. M. (1974) Nature 250,238-240. 14. Sheppard, H. & Burghardt, C. R. (1974) Res. Commun. Chem. Pathol. Pharmacol. 8,527-534. 15. Iversen, L. L., Horn, A. S. & Miller, R. J. (1975) in Pre- and Postsynaptic Receptors, eds. Usdin, E. & Bunney, W. E. (Dekker, New York), pp. 207-241. 16. Woodruff, G. N., Watling, K. J., Andrews, C. D., Poat, J. A. & McDermed, R. (1977) J. Pharm. Pharmacol. 29, 422-427. 17. Snyder, S. H., Creese, I. & Burt, D. R. (1975) Psychopharmacol. Commun. 1, 663-673. 18. Creese, I., Burt, D. R. & Snyder, S. H. (1976) Science 192, 481-483. 19. Leysen, J. & Laduron, P. (1977) Life Sci. 20, 281-288. 20. Pardo, J. V., Creese, I., Burt, D. B. & Snyder, S. H. (1977) Brain Res. 125, 376-382. 21. Burt, D. R., Creese, L. & Snyder, S. H. (1976) Mol. Pharmacol. 12,800-812. 22. Sokal, R. A. & Rohlf, F. J. (1969) Biometry (Freeman, San Francisco, CA), pp. 404-493. 23. Goldstein, A. (1964) Btostatistics: An Introductory Text (Macmillan, New York), pp. 129-187. 24. Woolf, C. M. (1968) Principles of Biometry (Van Nostrand, Princeton, NJ), pp. 288-293. 25. Klotz, I. M. & Hunston, D. L. (1971) Biochemistry 10, 30653069. 26. Vallner, J. J., Perrin, J. H. & Wold, S. (1976) J. Pharm. Sci. 65, 1182-1187. 27. Komiskey, H. L., Miller, D. D. & Patil, P. N. (1976) Pharmacologist 18, 134. 28. Cannon, J. G. (1975) Advances in Neurology 9, 177-183. 29. Cannon, J. G., Kim, J. C., Aleem, M. A. & Long, J. P. (1972) J.

Med. Chem. 15,348-350.

30. Schoenfeld, R. I., Neumeyer, J. L., Dafeldecker, W. & RofflerTarlov, S. (1975) Eur. J. Pharmacol. 30, 63-68. 31. Neumeyer, J. L., Dafeldecker, W. P., Costall, B. & Naylor, R. J.

(1977) J. Med. Chem. 20,190-196.

32. Costall, B., Naylor, R. J. & Cannon, J. G. (1977) Eur. J. Pharnacol. 41, 307-319.

Conformation of dopamine at the dopamine receptor.

Proc. Nati. Acad. Sci. USA Vol. 75, No. 6, pp. 2641-2643, June 1978 Biochemistry Conformation of dopamine at the dopamine receptor (conformationally...
591KB Sizes 0 Downloads 0 Views