Proc. Nati. Acad. Sci. USA Vol. 75, No. 12, pp. 5783-5787, December 1978 Biochemistry Selective labeling of serotonin receptors by d-[3H]lysergic acid diethylamide in calf caudate (ergots/hallucinogens/tryptamines/norepinephrine/dopamine) 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 catecholaminergic 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-adrenergic 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 phentolamine (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 spiperone (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 catechol- 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 apomorphine (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 dopaminergic 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 neurotransmitter 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 atropine sulfate, dihydroergotamine 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, methysergide hydrogen maleate, morphine 0 50 - sulfate, bufotenine monooxalate hydrate, 5,6-dihydroxytryp- III1111 1111 I 11 tamine creatinine sulfate, and mescaline 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, psilocin, bufotenine, and LSD. The fol- -J lowing drugs were also kindly donated: butaclamol (Ayerst I Research Laboratories); imipramine (Ciba-Geigy Canada, 60 Ltd.); fenfluramine (A. H. Robins Co.); metergoline (Farmi- 50 - I I- iiil11 I I1 iiil 111111 talia); mianserin (Organon Labs); quipazine (Miles Laboratories 100 Inc.), and methylsergide (Sandoz).