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Home , Apomorphine, Fenfluramine, Metergoline, Phentolamine, Propranolol, Spiperone

Proc. Nati. Acad. Sci. USA Vol. 75, No. 12, pp. 5783-5787, December 1978 Biochemistry Selective labeling of receptors by d-[3H] diethylamide in calf caudate (////) PATRICIA M. WHITAKER AND PHILIP SEEMAN* Department of Pharmacology, University of Toronto, Toronto, Canada M5S 1A8 Communicated by Philip Siekevltz, August 18,1978

ABSTRACT Since it was known that d-lysergic acid di- The objective in this present study was to improve the se- ethylamide (LSD) affected as well as sero- lectivity of [3H]LSD for serotonin receptors, concomitantly toninergic neurons, the objective in this study was to enhance using other drugs to block a- and dopamine receptors the selectivity of [3HJISD binding to serotonin receptors in vitro by using crude homogenates of calf caudate. In the presence of (cf. refs. 36-38). We then compared the potencies of various a combination of 50 nM each of (adde to pre- drugs on this selective [3H]LSD binding and compared these clude the binding of [3HJLSD to a-adrenoceptors), apmo ie, data to those for the high-affinity binding of [3H]serotonin and (added to preclude the binding of [3H[LSD to (39). dopamine receptors), it was found by Scatchard analysis that the total number of 3H sites went down to 300 fmol/mg, compared to 1100 fmol/mg in the absence of the - METHODS amine-blocking drugs. The IC50 values (concentrations to inhibit Preparation of Membranes. Calf brains were obtained fresh binding by 50%) for various drugs were tested on the binding of [3HLSD in the presence of 50 nM each of (A), from the Canada Packers Hunisett plant (Toronto). The phentolamine (P) and spiperone (S) With this combination, the caudates were removed within 2 hr after death, pooled, sliced IC50 for serotonin was 35 nM (compared to 1000 nM without it), into small cubes, and suspended in buffer at an approximate indicatin that 3HLSD had become considerably more selec- concentration of 50 mg wet weight per ml of buffer. (Caudates tively displaceable by serotonin under these conditions whereas were chosen because of the known presence of the effects of norepinephrine and dopaine on [ D binding were eliminated. Various ergots had aproximately equal IC50 and adrenergic as well as serotoninergic receptors, so that the values against [3H]serotonin and [L but tryptamines were specificity of our system could be tested.) The buffer contained much more selective against [3H serotonin; the data may indi- 15 mM Tris-HCI (pH 7.4), 5 mM Na2EDTA, 1.1 mM ascorbate, cate the existence of the two types of serotonin receptors. and 12.5 ,M nialamide. A preliminary crude homogenate of d-Lysergic acid diethylamide (LSD) has been extensively the suspension was made with a glass homogenizer and a Teflon studied since its discovery (1). The structural resemblance of piston (0.1.3-0.18 mm clearance); this piston, rotating at 500 serotonin (2) to LSD was subsequently noted, and evidence rpm, was passed up and down 10 times. The crude homogenate accumulated suggesting that LSD exerts its effects through the was incubated at 370C for 60 min and then stored in 5-ml ali- serotoninergic system (3-11). Convincing quots at -20°C for future use. Before use, the samples were evidence also indicates dopaminergic involvement in LSD thawed, resuspended with an additional 5 ml of buffer, and activity (12-19) as well as an a-adrenergic (20-22) and possibly centrifuged at 39,000 X g for 15 min at 4°C. The supernatant a histaminergic component (23). was discarded and the pellet was resuspended in 15 ml of buffer Although these findings suggest that LSD activity may not by using five up-and-down strokes of a ground-glass homoge- be limited to the serotoninergic system, high-affinity binding nizer. This suspension was then homogenized by a Polytron sites for [3H]LSD have been detected (24-29) and have been homogenizer (Brinkmann, Westbury, NY) at a setting of 7 (full suggested to label serotonin receptors (27, 28). The interpre- range = 10) for 10 sec, using a PT-10 homogenizer probe and tation of these early data is not clear because it is known that a 50-ml polycarbonate tube to contain the suspension. This the serotonin concentration that inhibits [3H]LSD binding by resulted in a final protein concentration of 0.2 mg per tube. 50% (IC5) is much greater than the concentration of serotonin Because the membrane homogenate was made up immediately that is known to produce a physiological effect. For example, prior to use, it was not necessary to store it on ice. although serotonin is known to have biological activity on pe- Serotonin-Specific [3HJLSD Binding Assays. The assays ripheral tissues at concentrations ranging from 1 to 100 nM were done by using 12 X 75 mm glass test tubes in which the (3-35), the reported IC50 values have varied from 200 to 2000 following aliquots were placed (Eppendorf Brinkmann pipettes nM (24-29). These apparent differences may not be pertinent, with polypropylene tips) for control binding: 0.1 ml of nonra- however, because the effective serotonin concentration for dioactive LSD (final concentration, 200 nM) or 0.1 ml of buffer; action on brain serotonin receptors is not known. Furthermore, 0.1 ml of buffer containing apomorphine, phentolamine, and it is possible that the serotonin receptors may have different spiperone (final concentration, 50 nM for each, referred to as preferred conformations which may require different serotonin the APS system); 0.2 ml of [3H]LSD (11.5 Ci/mmol; New En- concentrations to be effective. It is also possible that, in these gland Nuclear; final concentration, 2.0 nM); and 0.2 ml of brain early studies (24-29), serotonin may have competed with homogenate. For tubes with varying concentrations of drugs [3H]LSD for sites to which the serotonin might not normally competing against [3H]LSD, the final contents of the tubes were bind physiologically and which, therefore, might not have been the same as for control except that the 0.1 ml of buffer or non- serotonin receptors. radioactive LSD was replaced with 0.1 ml of drug solution.

The publication costs of this article were defrayed in part by page Abbreviations: LSD, d-lysergic acid diethylamide; IC50, concentration charge payment. This article must therefore be hereby marked "ad- that inhibits [3HJLSD binding by 50%; APS system, mixture containing vertisement" in accordance with 18 U. S. C. §1734 solely to indicate 50 nM each of apomorphine, phentolamine, and spiperone. this fact. * To whom reprint requests should be addressed. 5783 Downloaded by guest on September 25, 2021 5784 Biochemistry: Whitaker and Seeman Proc. Natl. Acad. Sci. USA 75 (1978) Each determination was done in quintuplicate, and each drug . was tested at eight or more different concentrations. * _ After the tubes were incubated, a 0.5-ml aliquot was removed from each tube and filtered.by vacuum through a glass fiber 80 filter (GF/B, Whatman, 24 mm diameter) on a Millipore stainless steel mesh support. The filter was then washed with 60 -A so o 'j 100I 1,000 100,000

delivered : --- -*--- __ was .**--o- wash o- The Np 5 ml of buffer at room temperature. ° ° by gravity from a syringe Repipette in <2 sec. The filter was 75 Dopamine, nM Aquasol (New placed into a liquid scintillation vial, 8 ml of 4.0 *-00100 England Nuclear) was added, and the filters were monitored 0 for [3H]LSD (after 6 hr, during which time the filters became . 80 translucent). Two types of LSD-displaceable binding were used: B defined as the total amount .0 (i) specific binding of [3H]LSD, .0 0) 1 10 100 1,000 10,000 100,00 bound minus that bound in the presence of 200 nM nonra- -6I00 Norepinephrine, nM dioactive LSD: and (fi) specific binding of [3H]LSD in the APS -i100 system, defined as the total amount of [3H]LSD bound in the I: presence of the APS system minus that bound in the presence of 200 nM nonradioactive LSD and the APS system Drugs. All drugs used in this study were HCI salts, except sulfate, tartrate, sodium diphe-

100 100 1,000 Serotonin, nM FIG. 2. Competition of dopamine (A), norepinephrine (B), and serotonin (C) against the total binding of [3HJLSD (- - 0 - -) and against the binding of [3H]LSD in the presence of the APS system (*--). In the presence of the APS system, the action of serotonin became much more selective (left shift), whereas dopamine and norepinephrine showed little competition against [3H]LSD (right shift). Specific binding of [3H]LSD (100%) was defined as that dis- placeable by 200 nM LSD. Specific binding of [3H]LSD in the pres- ence of APS was defined as that displaceable by 200 nM LSD in the rI IIiii iiil I. i B - presence of APS. Vertical bars indicate SEM for four to six experi- ments per point. .5 80

0 L) 60 nylhydantoin, hydrogen maleate, 0 50 - sulfate, bufotenine monooxalate hydrate, 5,6-dihydroxytryp- III1111 1111 I 11 tamine creatinine sulfate, and sulfate. We are ct0C grateful to the Health Protection Branch, Department of Na- 0 C .0 tional Health and Welfare, Canada, for supplying us with di- 80 methyltryptamine, , bufotenine, and LSD. The fol- -J lowing drugs were also kindly donated: (Ayerst I Research Laboratories); (Ciba-Geigy Canada, 60 Ltd.); (A. H. Robins Co.); (Farmi- 50 - I I- iiil11 I I1 iiil 111111 talia); (Organon Labs); (Miles Laboratories 100 Inc.), and methylsergide (Sandoz). D_

80 - RESULTS Conditions Selective for [3H]LSD Binding to Serotonin 60 Receptors. Total [3H]LSD binding, displaceable by excess time 50 - nonradioactive LSD, was first determined. The optimal for incubation was found to be 30 min, and the minimal con- 40 1 I I II I centration of excess nonradioactive LSD for total displacement 1 10 100 1000 nM was 200 nM. To preclude the binding of [3H]LSD to presynaptic and FIG. 1. Competition of phentolamine (A), spiperone (B), apo- postsynaptic dopamine receptors, competition by apomorphine morphine (C), and spiperone plus apomorphine (D) in the binding and spiperone was first studied. Fig. 1 shows that 50 nM of each of [3H]LSD to crude homogenate of calf caudate nucleus. In each of sites. the first three cases a plateau occurred in the 20-100 nM range, was sufficient to block [3H]LSD from these Similarly, suggesting that [3H1 LSD bound to high-affinity sites, some of which a-adrenergic sites were blocked by 50 nM phentolamine. were in common with phentolamine (a-adrenoceptors) and spiperone In order to test the increased sensitivity of our assay for se- and apomorphine (dopamine receptors). In D, the two drugs were rotonin sites, the competition curves for the three neuro- present at the same concentration. Data are shown as mean :SEM transmitters, dopamine, norepinephrine, and serotonin, without for four to six separate determinations, each in quintuplicate. Specific APS and with APS were compared (Fig. 2). For norepinephrine binding of [3H]LSD (100%) was defined as that displaceable by 200 nM LSD. and dopamine, the competition curves were shifted to the right Downloaded by guest on September 25, 2021 Biochemistry: Whitaker and Seeman Proc. Natl. Acad. Sci. USA 75 (1978) 5785 Table 1. Effect of various drugs on [3H]LSD binding in the presence of the APS system IC50, nM* : Acetylcholine NE Dopamine >100,000 Epinephrine NE 90 NE E E 'y-Aminobutyric acid C,.- Histamine NE 70 + Norepinephrine NE Serotoninergic drugs (-related): u- 50 Serotonin 35 Bufotenine 65 5-Methoxydimethyltryptamine 85 30- Psilocin 290 Tryptamine 500 10 50 N,N-Dimethyltryptamine 580 5,6-Dihydroxytryptamine 960 NE [3H]LSD bound, fmol/mg 5-Hydroxyindoleacetic acid NE Indole NE FIG. 3. Scatchard analysis of the binding of [3HJLSD to crude Melatonin NE homogenates ofcalf caudate. In the presence ofthe APS system, the derivatives: [3H]LSD sites were decreased from 1100 to 300 fmol/mg of protein. Dihydroergotamine 7 Specific binding of [3H]LSD was defined as that displaceable by 200 10 nM LSD. Specific binding of [3H]LSD in the presence of APS was LSD defined as that displaceable by 200 nM LSD in the presence ofAPS. Methysergide 22 Vertical bars indicate SEM for three to eight experiments, each point Metergoline 25 in quintuplicate; horizontal bars indicate the concentration range of Brom-LSD 50 points that were grouped together. 70 Serotonin antagonists and others: Methiothepin 8 in the presence of APS, showing a decreased sensitivity to these Mianserin 75 neurotransmitters, whereas for serotonin the curve was shifted Quipazine 950 to the left, indicating an enhanced selectivity of [3H]LSD for (+)-Butaclamol 80 serotonin sites in this assay. (-)-Butaclamol NE Properties of [3H1LSD Binding in the APS System. As shown in Fig. 3, a Scatchard analysis revealed approximately 850 1100 fmol of sites per mg for [3H]LSD without APS and almost Imipramine -1,000 300 fmol of sites per mg for [3H]LSD with APS. The Kd for 10,000 [3H]LSD without APS was 8.2 nM, and with APS it was 4.8 Nortryptiline 1,750 nM. »3,500 An important finding (Fig. 2) was that the IC50 for serotonin 3,500 against [3H]LSD was 35 nM, a value considerably lower than Amitryptiline 460 that reported by others (24-29). Because this IC50 value for Bromocryptine 230 serotonin had been obtained in the presence of the APS system, Additional compounds that had no effect at 3500 nM were atropine, , deanol, diphenylhydantoin, fenfluramine, , , , , mescaline, morphine, nialamide, and . * NE, no effect. -100 00 0 a further control experiment was done to ensure that these three 0 drugs had negligible effect on the serotonin ICso the ICso for serotonin in the presence of 100 nM apomorphine, spiperone, 60- and phentolamine was not significantly different from 35 nM. 0 o .040-6~ ~ ~ ~ ~ nQoipazine Competition of Various Drugs with [3HJLSD Binding (APS a-J System) Various serotonin , antagonists, neuro- 20- transmitters, ergots, and other drugs were tested for their ability ~~~~Methysergide - Silocin to compete against [3H]LSD in the APS system. Several exam- ples of such competition data are shown in Fig. 4 and the full 1 10 100 1,000 10,000 list of IC50 values is presented in Table 1. FIG. 4. Examples of results for drugs competing against the binding of [3H]LSD (in the presence of the APS system). The IC50 DISCUSSION values were derived from these types of experiments. Specific binding of [3HJLSD was defined as that displaceable by 200 nM LSD. Specific The data indicate that the selectivity of [3H]LSD for serotonin binding of [3HJLSD in the presence of APS was defined as that dis- sites was considerably enhanced in the presence of the APS placeable by 200 nM LSD in the presence of APS. Vertical bars in- system. This conclusion is supported by three main observations: dicate SEM for four experiments. (i) the competition graphs of bound [3H]LSD vs. dopamine or Downloaded by guest on September 25, 2021 5786 Biochemistry: Whitaker and Seeman Proc. Natl. Acad. Sci. USA 75 (1978) that the site(s) occluded by the APS must have a Kd somewhat higher than 8.2 nM; in order to detect this lower affinity site (as a separate component in the Scatchard graph), many more C~~~~~~~~ measurements would be needed. Generally, the order of potency of various drugs in displacing C locins Motergollgne. Tryptamine [3H]LSD (with APS) was similar to that found in our work on Methysergide /YMT 5-[3H1hydroxytryptamine binding (39). The two sets of data " LSD,/ *5-Methoxy-NN,- are correlated in Fig. 5. The tryptamine derivatives have ap- V Bufotenine dimethyltryptemine Dihydroergotamine proximately 'A0th the potency for the [3H]LSD site (with APS) compared to the 5-[3H1hydroxytryptamine site, whereas the ergot derivatives were of equal potency in both systems. ,Serotonin If it is assumed that the system is free of contamination by other neurotransmitter receptors, the relationships in Fig. 5 1 10 100 1000 suggest the possible existence of either two types of serotonin ICS,0 [3HJLSD (with APS), nM sites or possibly different sites for LSD and serotonin. The ergots (e.g., LSD) have a 10-fold higher preference for one of the two FIG. 5. Relationships between the IC50 values of various drugs these data cannot be for the binding of [3H]serotonin (data from ref. 39) and the IC50 values sites than do the tryptamines. However, of these same drugs for the binding of [3H]LSD (in the presence of the used to determine which set of sites are postsynaptic and which APS system; data from Table 1). The ergots have approximately equal are presynaptic. The IC50 ratio (i.e., 5-hydroxytryptamine potencies against both radioligands; the tryptamines have about IC50/LSD IC50) is about 1.0 for theergot but about 0.1 for the 10-fold higher potency against [3H]serotonin binding compared to tryptamines. These results are compatible with other findings [3H]LSD binding. on autoreceptor activity on indoles and LSD (40, 41). Finally, the data in Table 2 compare the IC50 values for were to the right in the APS system (Fig. [3H]LSD on the calf striatum (APS system) with those obtained norepinephrine shifted It can 2), showing a selective decrease in sensitivity to these neuro- by Bennett and Snyder (27) on rat frontal cortex tissue. transmitters; (it) the competition graph of [3H]LSD vs. serotonin be seen that the APS system enhances the selectivity of the se- was selectively shifted to the left in the APS system (Fig. 2), rotonin agonists for the [3H]LSD sites. showing an enhanced selectivity to this neurotransmitter; and We thank Mrs. Joan Dumas and Mrs. Anna Banaszuk for their ex- (tii) the IC50 of serotonin on [3H]LSD (with APS) was 35 nM. cellent technical assistance and Dr. Milt Titeler for his helpful sug- This value, 35 nM serotonin, shows much greater affinity for gestions and support. This work was supported by the Ontario Mental the [3H]LSD labeled sites than has been reported from previous Health Foundation and the Medical Research Council of Canada. studies wherein IC50 values for serotonin were 200 nM (24), 790 nM (25), 1900 nM (26), 1000 nM (27), or 500 nM (28). We in- 1. Stoll, W. A. (1947) Schweiz. Arch. Neurol. Psychiatr. 68,279- terpret these higher values obtained by others as stemming from 323. the competition between [3H]LSD and serotonin at serotonin 2. Twarog, B. M. & Page, I. H. (1953) Am. J. Physiol. 175,157- and nonserotonin sites. It is also possible that minor 161. technical differences (in the assays) between different workers 3. Woolley, D. W. & Shaw, E. (1954) Proc. Nati. Acad. Sci. USA may give different results, because it is known that this filtration 40,228-231. method is theoretically inferior to equilibrium dialysis (in which 4. Gaddum, J. H. (1957) Ann. N. Y. Acad. Sci. 66,643-648. desorption do not arise). 5. Costa, E. (1956) Soc. Exp. Biol. Med. 91,39-41. problems of radioligand 6. Welsh, J. H. (1957) Ann. N. Y. Acad. Sci. 66,618-630. The reduction in the number of [3H]LSD sites in the presence 7. Mansour, T. E., Sutherland, E. W., Rall, T. W. & Beudling, E. of APS (Fig. 3) apparently represents the nonserotoninergic (1960) J. Biol. Chem. 235,466-470. contribution of [3H]LSD binding which we have eliminated. 8. Freedman, D. X. & Giarman, N. J. (1962) Ann. N. Y. Acad. Sci. This nonserotoninergic site contributed approximately 800 96,98-107. fmol/mg to the total [3H]LSD binding of 1100 fmol/mg. 9. Anden, N.-E., Corrodi, H., Fuxe, K. & Hikfelt, T. (1968) Br. J. Clearly, this large site, if not eliminated, would Pharmacol. 34, 1-7. seriously decrease the usefulness of [3H]LSD as a for 10. Aghajanian, G. K., Foote, W. E. & Sheard, M. H. (1968) Science labeling serotonin sites. The Kd for [3H]LSD without APS was 161,706-708. 8.2 nM, whereas with APS it was 4.8 nM. Hence, this suggests 11. Goodwin, J. S., Katz, R. I. & Kopin, I. J. (1969) Nature (London) 221,556-557. .12. Pieri, L., Pieri, M. & Haefely, W. (1974) Nature (London) 252, 586-588. Table 2. Comparison of IC50 data for [3HJLSD (with APS) 13. Trulson, M. E., Stark, A. D. & Jacobs, B. L. (1977) Eur. J. Phar- Calf striatum Frontal cortex macol. 44, 113-119. (see Table 1), (ref. 27), 14. Menon, M. K., Clark, W. G. & Masouka, D. T. (1977) Psycho- nM nM pharmacology 52, 291-297. 15. Von Hungen, K., Roberts, S. & Hill, D. F. (1974) Nature (Lon- Methiothepin 8 60 don) 252,588-589. LSD 10 8 16. Sato, M. & Sawada, M. (1975) Neuropharmacology 14, 883- Methylsergide 22 100 886. Serotonin 35 1000 17. Christoph, G. R., Kuhn, D. H. & Jacobs, B. L. (1977) Proc. Soc. Brom-LSD 50 10 Neuroscs. 7,772. Bufotenine 65 300 18. Persson, S.-A. (1977) Eur. J. Pharmacol. 43,73-83. Mianserin 75 0oo 19. Meltzer, H. Y., Fessler, R. G., Simonovic, M., Doherty, J. & Fang, 1000 V. S. (1977) 54,39-44. Psilocin 290 20. Carino, M. A. & Horita, A. (1977) Life Sci. 20,49-56. Tryptamine 500 5000 21. Horita, A. & Hamilton, A. E. (1969) Science 164,78-79. Chlorpromazine 850 100 22. Bapna, J. S., Dandiya, P. C. & Kulkarni, S. K. (1973) Jpn. J. Haloperidol >>3500 2000 Pharmacol. 23, 735-737. Downloaded by guest on September 25, 2021 Biochemistry: Whitaker and Seeman Proc. Nati. Acad. Sct. USA 75 (1978) 5787

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