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Proc. Natl. Acad. Sci. USA Vol. 75, No. 5, pp. 2249-2253, May 1978 Biochemistry

Selective labeling of a- receptors in caudate nucleus by [3H] in the presence of spiperone-blocked receptors (/catechols/neuroleptics) M. TITELER AND P. SEEMAN Department of Pharmacology, University of Toronto, Medical Sciences Building, Toronto, Canada, M5S 1A8 Communicated by Charles H. Best, March 3, 1978

ABSTRACT Because it was known that [3H]dihydroergo- MATERIALS AND METHODS cryptine can label a-adrenergic receptors as well as dopa- mine receptors, this study was done to establish the conditions Preparation of Calf Caudate Homogenates. the experi- under which [3H]dihydroergocryptine would be a reliable ligand ments were done on crude homogenates of calf caudate, pre- for selective labeling of a-adrenergic receptors. The calf caudate pared as described. (12). In order to retain most of the dopamine was chosen because it contains both dopamine and adrenergic receptors, the homogenates were not purified or subfraction- receptors, and 5 nM spiperone (spiroperidol) was used to block ated. Calf brains were obtained fresh from the Canada Packers the neurole tic dopamine receptors. Thus, in the presence of Hunnisett plant (Toronto, Canada). The caudates were re- spiperone, [ Hdihydroergocryptine exhibited saturable binding moved within 2 hr after with a Kd of 0.73 nM and a total number of sites of 150 fmol/mg death, pooled, sliced into small cubes, of protein. The catechol neurotransmitters competed for [3HJ and suspended in buffer at an approximate concentration of dihydroergocryptine binding in the potencies order epinephrine 50 mg (wet weight)/ml of buffer (15 mM Tris-HCl, pH 7.4/5 > (-)norepinephrine > dopamine, indicating that [3HJdihy- mM Na2EDTA/1.1 mM ascorbate/12.5 ,uM nialamide). A droergocryptine (in the presence of 5 nM spiperone) was re- preliminary crude homogenate of the suspension was made by vealing a receptors. The a-adrenergic antagonists also competed using a glass homogenizer with a Teflon piston (0.13-0.18 mm for binding in the appropriate order: > phen- clearance). This piston, rotating at 500 rpm, was passed up and oxybenzamine > dibenamine. Finally, was down 20 times in the homogenizer. The crude homogenate was more potent than in competing for [3H]dihydroer- first incubated at for 60 min and gocryptine, also in accord with the properties of a receptors. 370 then stored in 3-ml ali- These results with [3H]dihydroergocryptine as an a-adrenergic quots at -20° for future use. Before use, the samples were receptor ligand correlate well with those published by others thawed, resuspended in the glass/Teflon homogenizer and for [3H]WB4101. homogenized by hand (10 up-and-down passes), and centri- fuged at 39,000 X g for 15 min at 40; the supernatant was dis- Several radioactive substances have been used in the past few carded and the pellet was resuspended in 10 ml of buffer. The years to label the a- in mammalian brain suspension was finally homogenized with a Polytron homoge- homogenates. These include [3H]dihydroergocryptine (1-6), nizer (Brinkmann Instrument Co.) at a setting of 7 (full range [3H] (7, 8), [3H]WB-4101 (7-9), [3H]norepinephrine = 10) for 20 sec, using a PT-10 homogenizer probe and a 50-ml (10, 11), and [3H]epinephrine (10, 11). It has been found, polycarbonate tube to contain the suspension. Homogenization however, that [3H]dihydroergocryptine can also identify at settings higher than 7 led to a loss of protein through the glass dopamine (12-14) and possibly (2, 15) receptor sites. fiber GF/B filters used in the radioreceptor assays. Except In fact, [3H]dihydroergocryptine (at 0.7 nM) becomes selective where indicated, the homogenates were always kept chilled on for dopamine receptors in the presence of an excess (500 nM) ice. of phentolamine to block the a-adrenergic receptors (12). [3H]Dihydroergocryptine Binding Assays. The [3H]dihy- The present study was done in order to improve the selective droergocryptine was purchased from New England Nuclear labeling of a-adrenergic receptors by [3H]dihydroergocryptine Corp. (Boston, MA) and was used without further purification; in the calf caudate nucleus. The the specific activity was 21 Ci/mmol. The material was stored caudate nucleus contains many in ethanol at -20°. dopamine receptors and it was necessary to determine that The [3H]dihydroergocryptine could, in fact, properly label a-ad- [3H]dihydroergocryptine binding assays were done in renergic glass test tubes (12 X 75 mm), in which the following aliquots receptors in the presence of a dopamine-receptor were placed (using Eppendorff-Brinkmann pipettes with blocking drug. polypropylene tips): 0.2 ml of [3H]dihydroergocryptine (final Of the a-adrenergic radioligands now available, it appears concentration, 0.2-0.7 nM, but with the majority at 0.7 nM); that [3H]WB-4101 is the one that best meets the appropriate 0.2 ml of brain homogenate (always added last and containing criteria (see Discussion). A second goal of this study, therefore, between 0.2 and 0.3 mg of protein); and 0.2 ml of buffer, 0.1 was to test whether the binding properties of [3H]dihydroer- ml of buffer and 0.1 ml of drug, or 0.1 ml of drug and 0.1 ml gocryptine to a-adrenergic receptors would match those of of another drug. Each determination was always done in [3H]WB-4101 and thus provide a second and different type of sextuplicate. After the samples were incubated for 60 min (220), radioligand for a-adrenergic receptors. a 0.5-ml aliquot was removed (polypropylene pipette tip) from the mixture and filtered under reduced pressure through a glass The costs of publication of this article were defrayed in part by the fiber filter (GF/B; Whatman; 24 mm diameter) on a Millipore payment of page charges. This article must therefore be hereby marked stainless steel mesh support; the filtration took less than 1 sec. "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate The filter was then washed twice with 7.5 ml of buffer per this fact. wash. The wash buffer was delivered by gravity from a syringe 2249 Downloaded by guest on September 24, 2021 2250 Biochemistry: Titeler and Seeman Proc. Natl. Acad. Sci. USA 75 (1978)

111 111 I ' I' III,, III binding is in accordance with a high affinity of spiperone for the neuroleptic/ (Kd 0.5 nM) and a much 100 lower affinity for the a-adrenergic receptor (Kd 50 nM), as indicated by Peroutka et al. (9) who used [3H]WB-4101. The 'O 80 _ results in Fig. 1 indicate that 5 nM spiperone would saturate the 30 dopamine receptor and leave the a-adrenergic receptor free .0 for interaction with 0.7 nM [3H]dihydroergocryptine. 60_so The specific binding of [3H]dihydroergocryptine over a I range of [3H]dihydroergocryptine concentrations in both the I 40 presence and the absence of 5 nM spiperone is shown in Fig. 2. The definition of nonspecific binding of [3H]dihydroergo- cryptine in the absence of spiperone was that amount of [3H]- 20 dihydroergocryptine bound in the presence of 500 nM (+)- , which has been shown to saturate both a-adren- ol uuuuuuI ergic and dopamine receptors (9, 12). The definition of non- 0.1 1.0 10 100 specific binding of [3H]dihydroergocryptine in the presence Spiperone, nM of 5 nM spiperone was that amount of [3H]dihydroergocryptine FIG. 1. Biphasic action of spiperone on the binding of [3H]- bound in the presence of 500 nM phentolamine. Thus, spip- dihydroergocryptine ([3H]DHE) to calfcaudate particulate. The ra- dioligand concentration was 0.7 nM. Data are shown as means + erone decreased the total binding of [3H]dihydroergocryptine SEM. from 328 fmol/mg to 150 fmol/mg, presumably due to blockade of dopamine receptors. Re-pipette over a period of 4 sec. The filters were not blotted Effect of Neurotransmitters on Specific [3HJDihydroer- or dried but were placed directly into liquid scintillation vials, gocryptine Binding in the Presence of 5 nM Spiperone. Fig. 8 ml of Aquasol (New England Nuclear Corp.) was added, and 3 and Table 1 illustrate the competition between [3H]dihy- the samples were assayed for 3H (42% efficiency) after storage droergocryptine and various neurotransmitters. The order of at 4° for at least 6 hr to allow temperature equilibration and to potency [epinephrine > (-)-norepinephrine > permit the glass fiber filters to become uniformly translu- > dopamine] indicates that an a-adrenergic receptor is being cent. identified (7-9). Effect of a-Adrenergic Antagonists on Specific [3H- RESULTS Dihydroergocryptine Binding (in the Presence of 5 nM Spi- Properties of Specific [3HJDihydroergocryptine Binding. perone). The competition of various a-adrenergic antagonists As we previously demonstrated (12), in order to use (3H]dihy- for [3H]dihydroergocryptine is shown in Fig. 4. Dihydroergo- droergocryptine as a specific ligand for a receptor, other re- cryptine, phentolamine, and strongly in- ceptors for which [3H]dihydroergocryptine has high affinity hibited at concentrations between 1 and 100 nM. Phenoxy- must be blocked by a suitable agent. In this case spiperone was benzamine was about 3-4 times more potent than dibenamine. chosen to block the dopamine receptor (Fig. 1) because this is These results are in accord with the pharmacological properties one of the most potent antidopaminergic drugs. The biphasic of the a-adrenergic receptor (9, 16, 17). competition curve of spiperone on [3H]dihydroergocryptine Effect of Neuroleptics on [3HjDihydroergocryptine

180 544

160 48(

,C 140 424 0

120 - 364 E

E 100 U. w~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-

[3HIDHE, nM B, fmol/mg FIG. 2. (Left) Specific binding of [3H]dihydroergocryptine to calf caudate membranes in the presence and absence of 5 nM spiperone. The definition of nonspecific binding of {3H]dihydroergocryptine in the absence of spiperone was that amount of [3H]dihydroergocryptine bound in the presence of 500 nM (+)-butaclamol which has been shown to saturate both a-adrenergic and dopamine receptors (9, 12). The definition of nonspecific binding of [3H]dihydroergocryptine in the presence of 5 nM spiperone was that amount of [3H]dihydroergocryptine bound in the presence of 500 nM phentolamine. (Right) Scatchard analysis of [3H]dihydroergocryptine binding indicates a dissociation constant (Kd) of 0.73 nM for [3H]dihydroergocryptine to the binding sites (in the presence of spiperone). Units for B/F: (fmol/mg)/(nmol/liter). Downloaded by guest on September 24, 2021 Biochemistry: Titeler and Seeman Proc. Natl. Acad. Sci. USA 75 (1978) 2251 Table 1. Inhibition of specific [3H]dihydroergocryptine binding in the presence of 5 nM spiperone Inhibitor Ki, nM

'0 a-Agonists: c Clonidine 40 0 .0 Epinephrine 90 (-)-Norepinephrine 260 LU; Phenylephrine 600 I a-Antagonists: I Dihydroergocryptine 0.25 Phentolamine 6 Phenoxybenzamine 12 Dibenamine 50 Neuroleptics: a-Flupenthixol 7 10 100 1,000 10.000 (+)-Butaclamol 7 Neurotransmitter, nM 8 FIG. 3. Effect of neurotransmitters [epinephrine, (-)-norepi- Chlorpromazine 12 nephrine and dopamine] on the specific binding of0.7 nM [3H]dihy- Spiperone 20 droergocryptine ([3H]DHE) (in the presence of 5 nM spiperone) to 30 calf caudate membranes. The order of potency, epinephrine > (-)- 34 norepinephrine > dopamine, is characteristic of an a-adrenergic re- Haloperidol 50 ceptor. All test tubes contained 5 nM spiperone in order to block dopamine receptors. Nonspecific binding of [3H]dihydroergocryptine Others: was defined as that occurring in the presence of both 5 nM spiperone Dopamine 1,000 and 500 nM phentolamine. 2,000 Isoproterenol 5,000 Binding (in the Presence of 5 nM Spiperone) Fig. 5 illustrates Serotonin 5,000 the potency of various neuroleptics in competing for [3H]- Bicucculline 7,000 dihydroergocryptine binding in the presence of 5 nM spiperone. (+)-Norepinephrine 10,000 The order of potency (chlorpromazine > spiperone or halop- 80,000 eridol) is characteristic of a-adrenergic receptors rather than Equilibrium constants of drugs and neurotransmitters as deter- dopamine receptors (refs. 9 and 17, pp. 170-171). mined by competitive inhibition of [3H]dihydroergocryptine binding Fig. 6 illustrates the correlation found when various a-ad- in the presence of 5 nM spiperone to calf caudate particulate. Con- renergic agonists and antagonists competed with either [3H]- ditions as in Fig. 3. The values were calculated from the ICWo values WB-4101 or [3H]dihydroergocryptine (in the presence of 5 nM (concentration ofdrug inhibiting specific binding by 50%) using the spiperone). This correlation indicates that [3H]WB-4101 and equation Ki = IC5d[1 + [A*]/Kd*J where A* is the concentration of [3H]dihydroergocryptine (in this study) are probably labeling radioligand and Kd* is the dissociation constant of the radioligand the same receptors. for the receptor (18). In Fig. 7 the relative potencies of neuroleptics for the a- adrenergic receptor and the dopamine receptor are correlated should have high affinity for the receptor, with haloperidol less in two ways. The ordinate represents the relative potencies of potent than chlorpromazine (refs. 9 and 17, pp. 170-171), which neuroleptics for the a-adrenergic and dopamine receptors is the opposite from the dopamine receptor (19, 20). All the determined with [8H]dihydroergocryptine. The abscissa rep- resents the same values with [2H]WB-4101 used to label the a-adrenergic receptor and [3H]haloperidol used to label the dopamine receptor. The striking correlation indicates that [3H]dihydroergocryptine does specifically label the a-receptor Phenoxybenzamine (in the presence of 5 nM spiperone) and the dopamine receptor (in the presence of 500 nM phentolamine). Table 1 lists the Ki values determined from the IC50 values obtained for various -; 60 neurotransmitters and drugs. The IC50 values were converted Phentolamine to Ki values by the equation Ki = CG50/[1 + (A*/Kd*)] (18), in 40 Dibenamine which A* is the total 3H-labeled ligand concentration and Kd* I Dihydro- \ is the equilibrium dissociation constant of the receptor for the 20 ergocryptine \ ligand. 3H-labeled 01 0.1 1.0 10 100 1,000 10,000 DISCUSSION Drug, nM In defining specific binding to the a-adrenergic receptor, FIG. 4. Effect of a-adrenergic antagonists (dihydroergocryptine, several pharmacological criteria must be met. Epinephrine phentolamine, phenoxybenzamine, and dibenamine) on the specific should be more potent than (-)-norepinephrine which in turn binding of0.7 nM [3H]dihydroergocryptine ([3H]DHE) (in the pres- should be more potent ence of 5 nM spiperone) to calfcaudate particulate. The high affinity than dopamine and serotonin. Phento- for phentolamine and phenoxybenzamine is characteristic of a-ad- lamine and phenoxybenzamine should be potent displacing renergic receptors. The 4-fold higher potency of phenoxybenzamine, agents, and phenoxybenzamine should be more potent than compared to phentolamine, is also indicative of an a-adrenergic re- dibenamine (ref. 17, pp. 533-544). Dopamine antagonists ceptor. Other conditions were as in Fig. 3. Downloaded by guest on September 24, 2021 2252 Biochemistry: Titeler and Seeman Proc. Natl. Acad. Sci. USA 75 (1978)

Spiperone 100 Pimozide * ic

C .E ctm 0 .CI c Haloperidol F m Q ° 10 9 /-ck~~~~~Flupenthixol

a, ._ a (+)-Butaclamol UPaL I w I I aNe 0 a

I I I 1 _n 1.0 ._ */Chlorpromazine 44 IL Promazine 0Clozapine

.I... ,a....I.,,..1 ,, 0.1 1.0 10 100 Ki( 3H]WB-4101)

Drug, nM Ki( [3HI Haloperidol)

FIG. 5. Effect of neuroleptic drugs on the specific binding of 0.7 FIG. 7. The ratio of Ki values may serve as an index of the relative nM [3H]dihydroergocryptine ([3H]DHE) (in the presence of 5 nM selectivity of the neuroleptic for a-adrenergic or dopamine receptors. spiperone) to calf caudate particulate. All these drugs were potent in The selectivity ratios with [3H]dihydroergocryptine correlate well with competing with [3H]dihydroergocryptine for binding. The order of the ratios with [3H]WB-4101 and [3H]haloperidol (9). potency, chlorpromazine > haloperidol, is again characteristic of an a-adrenergic receptor, and opposite to their relative potencies on ergocryptine has equal affinity for the dopamine receptor and dopamine receptors. Other conditions were as in Fig. 3. the a-adrenergic receptor (12). The only other ligand to fulfill all these criteria is [3H]WB- above criteria have been fulfilled by the use of [3H]dihydro- 4101 (7-9). This tritiated ligand.displayed the expected order ergocryptine in the presence of 5 nM spiperone to label the a-adrenergic receptor. of potency for neurotransmitters, a-antagonists, and neuroleptic agents. [3H]epinephrine, and [3H]cloni- These results thus provide a clear indication that [3H]dihy- [3H]Norepinephrine, dine did not fulfill all of the necessary criteria. For droergocryptine can properly label the a-adrenergic receptors apparently and were required at high in mammalian brain, providing that the dopamine sites are example, chlorpromazine haloperidol blocked. Davis et al. (ref. 2; cf. ref. 15) found serotonin to be concentrations to inhibit the binding of [3H]norepinephrine, or (7, 8, 10, 11), unlike the low more potent than the other neurotransmitters in competing for [3H]epinephrine, [3H]clonidine the binding sites in rat cerebral cortex, concentrations required by these two neuroleptics to inhibit the [3H~dihydroergocryptine of 10, 11). It has further been thus indicating an involvement with the serotonin receptor. binding [3H]WB-4101 (7, 8, that [3H]epi- Greenberg et al. (8) were apparently labeling both a-adrenergic suggested (7, 8, 10, 11) [3H]norepinephrine, and dopamine receptors in the caudate because [3H]dihydro- nephrine, and [3H]clonidine label the "agonist conformation" of the a-adrenergic receptor in order to explain why a-antag- onists concentrations to compete with ' ' '"H "1 I' '1'" I1"' require relatively high these three ligands. Phentolamine, however, was found to have 10-6 Phenylephrine high potency for both [3H]WB-4101 and the sites labeled by [3H]norepinephrine, [3H]epinephrine, and [3H]clonidine (10, (-)-Norepinephrine * am 11). It was further proposed that phentolamine is a partial an- tagonist, based on those binding data (10, 11). There is no evi-

* dence, however, of any but a full antagonist action of phento- E 10-' Epinephrine lamine at the a-adrenergic receptor (ref. 17, pp 533-544). In W / Dibenamine addition, the two-state concept (agonist and antagonist con- I Phenoxbenzae formation states) is not really necessary to explain the data for the dopamine receptor (refs. 12 and 21; unpublished data). that in studies aimed at the 10-8 We suggest, therefore, labeling O-P hentolamine a-adrenergic receptor and the dopamine receptor (19-21; unpublished data) there are two sets of sites being labeled. In the case of the a-adrenergic receptor, [3H]WB-4101 and [3H]dihydroergocryptine (in the presence of. 5 nM spiperone) l H l ,1 I[ are the main 10-9 10-8 1io-7 10o6 10o5 labeling pharmacological receptor. [3H]Norepi- and may be Ki([3HlWB 4101), mol/liter nephrine, [3H]epinephrine, [3H]clonidine labeling a-site different from the classical receptor. This other site may FIG. 6. Correlation of the equilibrium dissociation constants (KM) be a presynaptic (22, 23) site. and as determined by of various a-adrenergic agonists antagonists We thank Mrs. Carla Ulpian for her excellent technical assistance. the use of [3H]WB-4101 (9) and [3H]dihydroergocryptine (in the was the Ontario Mental Health presence of 5 nM spiperone). The Ki values for the catecholamines This work supported by Foundation, Foun- determined with [3H]WB-4101, were revised from the original data the Medical Research Council of Canada, and the Connaught to account for the differences in temperature. dation. Downloaded by guest on September 24, 2021 Biochemistry: Titeler and Seeman Proc. Nati. Acad. Sci. USA 75 (1978) 2253

1. Williams, L. T. & Lefkowitz, R. J. (1976) Science 192, 791- Sci. USA 74,3750-3753. 793. 13. Caron, M. G., Raymond, V., Lefkowitz, R. J. & Labrie, F. (1976) 2. Davis, J. N., Strittmatter, W., Hoyler, E. & Lefkowitz, R. J. -(1977) Fed. Proc. Fed. Am. Soc. Exp. Biol. 36,278. Brain Res. 132, 327-36. 14. Caron, M. G., Drouin, J., Raymond, V., Kelly, P. A. & Labrie, F. 3. Williams, L. T., Mullikin, D. & Lefkowitz, R. J. (1976) J. Biol. (1976) Clin. Res. 24, 656A. Chem. 251, 6915-6923. 15. Closse, A. & Hauser, D. (1976) Life Sci. 19, 1851-1864. 4. Williams, L. T. & Lefkowitz, R. J. (1977) Mol. Pharmnacol. 13, 16. Ariens, E. (1967) Ann. N.Y. Acad. 304-313. J. Sci. 139,624. 5. Strittmatter, W. J., Davis, J. N. & Lefkowitz, R. J. (1977) J. Biol. 17. Nickerson, M. & Collier, B. (1975) in The Pharmacological Basis Chem. 252, 5472-5477. of Therapeutics, eds. Goodman, L. S. & Gilman, A. (Macmillan, 6. Greenberg, D. A. & Snyder, S. H. (1977) Life Sci. 20, 927- Toronto), pp. 533-544, p. 170-171. 932. 18. Cheng, Y. C. & Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 7. U'Prichard, D. C., Greenberg, D. A. & Snyder, S. H. (1977) Mol. 3099-3108. Pharmacol. 13, 454-473. 19. Seeman, P., Chau-Wong, M., Tedesco, J. & Wong, K. (1975) Proc. 8. Greenberg, D. A., U'Prichard, D. C. & Snyder, S. H. (1976) Life Nati. Acad. Sci. USA 72,4376-4380. Sci. 19, 69-76. 20. Burt, D. R., Enna, S. J., Creese, I. & Snyder, S. H. (1975) Proc. 9. Peroutka, S. J., U'Prichard, D. C., Greenberg, D. A. & Snyder, Natl. Acad. Sci. USA 72,4655-4659. S. H. (1977) Neuropharmacology 16,549-556. 10. U'Prichard, D. C. & Snyder, S. H. (1977) Life Sci. 20, 527- 21. Titeler, M., & Seeman, P. (1978) in Dopamine, eds. Horn, A., Korf, 534. J. & Westerink, B. (Academic, London), in press. 11. U'Prichard, D. C. & Snyder, S. H. (1977) J. Biol. Chem. 252, 22. Langer, S. Z. (1974) Biochem. Pharmacol. 23,1793-1800. 6450-6463. 23. Werner, U., Starke, K. & Schumann, H. I. (1976) Arch. Int. 12. Tittler, M., Weinreich, P. & Seeman, P. (1977) Proc. Nat. Acad. Pharmacodyn. 195, 291-308. Downloaded by guest on September 24, 2021