323

European Jottrttat ofP/tarmacoiofiy, 205 (1991) 323-324 Q 1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/91/$03.50

E.JP 0324R

Rzpid communication

Cathy D. Mahle

‘, Henry P. Nowak

‘, Ronald J. Mattson

*, Stephen

D. Hurt

3, and Frank D. Yocca

I

CNS ’ Neuropharmacology and ’ Cttemistry Bristol-Myers Squibb Pharmaceutical Researctt Institute. Wallbtgford, CT 06492, U.S.A. and .’ New England Nuclear Products, 549 Albany Street, Boston, hf.4 02118, U.S.A.

Received 7 November 1991. accepted 8 November 1991

Radioligand binding studies in mammalian brain have demonstrated the existence of distinct populations of sites related to serotonin receptor subtypes (Frazer et al., 1990). For example, elimination of binding to 5-HT,, and SHT,, receptors has uncovered sites displaying high (SHT,,) and low (SHT,,) affinity for 5-carboxamidotryptamine (5-CT, Leonhardt et al., 1989). To probe the possible heterogeneity in sites related to 5-HT,o receptors, we have eliminated a portion of the complex nature of [3H15-HT binding by utilizing [‘HIS-CT. In the present study we demonstrate that under conditions which favor the binding of f3H]5-HT to 5-HT,, sites (Heuring and Peroutka, 19871, [3H]5-CT labels with high affinity at least two populations of binding sites in guinea pig striatum and frontal cortex. Guinea pig striatum and frontal cortex (Pel-Freeze Biologicals, Rogers, Arizona), were homogenized (50 mM HEPES/Tris, 2.5 mM MgCl,, and 2.0 mM EGTA, pH 7.41, washed twice (39000 x g, 10 min, 4”C), incubated at 37°C (10 min), recentrifuged and pellets resuspended in the original volume of buffer containing 0.1% ascorbic acid and 10 PM pargyline. Binding assays (60 min, room temperature) consisted of 0.5 nM [3H]5-HT or [“HIS-CT (specific activity 27-32 Ci/mmol), 100 nM 8-OH-DPAT and mesulergine, to inhibit [3H]5-CT binding to 5-HT,, or 5-HT,, receptors (Heuring and Peroutka, 1987) and competing drugs. Non-specific binding was defined by 10 PM 5-HT. [3H]5-CT (specific binding approximately 95%) was shown to label a slightly lower density (lo-15%) of binding sites compared to [3H]5-HT in bovine and guinea pig striatum (not shown).

Correspondence to: F.D. Yocca, CNS Neuropharmacology. Department 404. Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT 06492, U.S.A. Tel. 1.203.284 6357, fax 1.203.284 1711.

Figure 1 illustrates the pharmacological differences [‘HIS-HT and [3H]5-CT binding in guinea pig brain. 5-HT competed for [3H]5-HT in a monophasic

between

Fig. 1. Inhibition of [3H]S-HT (0.5 nM, A,B) and [3Hj_5-CT(0.5 nM. C,D) specific binding by 5-CT. 5-HT and sumatriptan in homogenates of guinea pig striatum and frontal cortex. Data shown are from a single experiment performed in duplicate, with standard deviation less than 58. Each ex;reriment was repeated an additional two to four times yielding similar results. For the illustrated expe;iment, analysis (LIGAND, accepted level of significance P G 0.01) revealed that in striatum and frontal cortex, 5-HT competed in a monophasic manner for [3H]5-HT (slope indexes 1.01 and 0.93, respectively) resulting in K, values (nM) 1.27 and 1.02 respectively. while both 5-CT and sumatriptan competed in a biphasic manner (slope indexes: striatum, 0.34, 0.59 and frontal cortex, 0.39, 0.37) resulting in two components (K,. nM): 5-CT 0.36 (64%) and 215 (36%), 1.77 (53%) and 505 (47%); sumatriptan 6.7 (66%) and 245 (34’%), 21 (51%) and 3608 (49%) (striatum and frontal cortex, respectively). Analysis of [‘H]5-CT competitions in striatum revealed 5-m and 5-HT competed in a monophasic manner (slope indexes 0.91 and 0.97, respectively) resulting in Ki values 0.61 and 1.21 nM, respectively. while sumatriptan competed in a biphasic manner (slope index 0.76). resulting in two components (Ki, nM) 27 (73%) and 400 (27%). In frontal cortex, S-CT competed in a monophasic manner (slope index 0.86) resulting in a K, of 2 nM. 5-HT and sumatriptan competitions were biphasic (slope indexes 0.71 and 0.37, respectively! resulting in two components (Ki, nM): 5-HT 4 (90%) and 246 (1OQ). sumatriptan: 41 (51%) and 6000 (49%).

th striatum and frontal cortex (K, 1.77 + k t1.99 nM, respectively). tlowever. both produced biphasic displacement in striatnm (K, S-CT O.,i;t rf:0.01. 190 & 36 .O 5 0.4. _U3 -+ 138 nM) and frontal es [K, S-CT 1.33 I 0.62. 407 f 139 nM, sumatript 2S3 nhi), resulting in a high Gd low aftimty GHT,E) component. When ration of [‘HI.%CT is utilized in competiid binding to S-HT,, sites. S-CT competed in a monophasic manner in striatum and frc!nFal cortex (K, 0.68 + 0.04. I.% + 0.35 nM, respecrive[v). S-I-IT produced monophasic inhibition of [‘HISCT binding in striatum (K, 1.43 + 0.1 I nM) but yielded biphasic dispfacement in frontal cortex. consisting of a similar high affmity component to that found in striaturn (EC, 4.0 1: 0.35 niM) representing approximately 87% of total binding, and an additional low affinity component (K, 0.43 i 0.22 PM) which comprised only 13% of total binding. Interestingly. sumsiriptan was abie to further discriminate amongst high affinity [“HE-CT sites. producing shallow displacement curves in both striatum and frontal cortex, where approximately 77% of the binding in striatum was high (Ki 18 f 3 nMJ and 23% low affinity (K, 1.2 + 0.7 PM). In frontal cortex. 48% possessed high affinity (K, 34 k 7 nM) while 52% exhibited a significantly lower affinity (Ki 3.5 i 0.7s FM). The high affinity and selectivity of [‘HIS-CT makes it an appropriate radioligand to investigate the tentative diversity in S-HT,_,ikr binding sites. This seems warranted since previously reported binding affinities have demonstrated clear discrepancies with the cloned human N-IT,, receptor (Hambiin and Metcalf, 1991). suggesting the existence of more than one type of S-HT,, receptor. Although the S-HT,, receptor is

linked to an inhibitory G-protein (Hamblin and Metcalf. 1991) and may exist in multiple affinity states (Waeber and Palarios. 19911, the low concentration of [‘HIS-CT (0.5 nM) used in this study would probably not occupy a significant portion of low affinity receptors. In the present communication, we have demonstrated that under conditions favoring occupancy of S-HT,. receptor sites, S-CT binds to at least three distinct sites: two with high affinity 6HT,,-like) and one with low affinity 6HT,,). Multiple S-HT,,-like binding sites distributed in a regionally disproportionate manner in guinea pig brain may suggest the existence of a similar phenomenon in human brain.

Acknowledgement The authors thank Dr. Saul Maayani for many stimulating discussions and critical review of the manuscript.

References Frazer, A.. S. Maayani and B.B. Wolfe, 1990, Subtypes of receptors for serotonin. Ann. Rev. Pharrnacol. Toxicol. 30, 307. Ilamblin. M.W. and M.A. Metcalf, 1991. Primary structure and functioraal characterization of a human 5-HT,o-type serotonin receptor. Mol. Pharmacol. 40, 143. Heuring, R.E. and S.J. Peroutka. 1987, Characterization of a novel “H-5-hydroxytryptamine binding site subtype in bovine brain membranes. J. Neurosci. 7, 894. Leonhardt. S.. K. Herrick-Davis and M. Titeler. 1989, Detection of a novel serotonin receptor subtype (S-HT,,) in human brain: interaction with a GTP-binding protein, J. Neurochem. 53, 465. Waeber. C. and J.M. Palacios, 1991, Serotonin: Molecular Biology, Receptors, and Functional Effects (BirkIuser Verlag, Basel) p. 107.