Application of [‘251]Iodocyanopindolol to Measure 5-HydroxytryptamineB Receptors in the Brain of the Rat’

In this study, [1251]iodocyanopindolol ([125I]ICYP), in the presence of isoproterenol, was used to label 5-hydroxytryptaminelB (5HTIB) receptors in homogenates of the cortex, substantia nigra and caudate-putamen of the rat. The determination of the appropnate concentrations of isoproterenol required to block optimally beta adrenoceptors whereas producing minimal occupancy of 5HT1B receptors was achieved by generating isotherms for isoproterenol at multiple concentrations of [125l]ICYP. When different concentrations of isoproterenol were used with increasing concentrations of [1251]ICYP, a linear Scatchard transformation of the saturation curve was achieved, even with ligand concentrations about 6-fold greater than the K0 for [125l]ICYP. Competition for [125IJICYP (1 00 pM)labeled binding sites by 1 5 serotonin agonists or antagonists was adequately described by a single site model, and the affinity of these drugs for the site labeled by [125l]ICYP was similar to that determined previously when using indirect methods to label 5-HT1B receptors. Serotonin itself showed high affinity for this binding site as did two antagonists, metergoline and methiothepin. By contrast, drugs thought to be selective for the 5-HT1A receptor (e.g. , 8-hydroxy-2-(di-n-propylamino)tetralin, buspirone and spiperone) showed very weak affinity for the binding site labeled with [125l]ICYP. The effect of nucleotide regulation on [1251]ICYP binding at 5-HT1B receptors also was evaluated. It was determined that GTP had little effect on the binding of [125IJICYP, reducing total binding by only 15% and shifting the displacement curve of 5-HT by a factor of less than two. The regulation of 5-HT1B receptors, labeled by [1251] ICYP, also was evaluated. Intraventricular injections of 5,7dihydroxytryptamine increased significantly the number of 5-HT1B receptors in the caudate-putamen; this treatment had no effect on 5-HT1B binding sites either in the cortex or substantia nigra. The regulatable binding sitefor [125l]ICYP in the caudate-putamen had a pharmacological profile very similar to that of the 5-HT1B binding site in the cortex. [125I]ICYP appears to be a useful ligand to measure 5-HT1B receptors in the brain of the rat. The localized increase in 5-HT15 receptors in the caudate-putamen after destruction of central serotonergic neurons might indicate that the majority of 5-HT1B receptors in this area of brain are not located on serotonergic nerve terminals. Pedigo et aL (1981) proposed originally that more than one subtype of a receptor for 5-HT, termed the 5-HT1 receptor, existed in the CNS of the rat. This hypothesis was based upon the ability of spiperone to inhibit the binding of [3H]-5-HT in a biphasic manner. The site for which spiperone had high affinity was termed the 5-HT1A subtype and the site for which it had low affinity was called the 5-HT1B subtype. More recently, a third 5-HT1 receptor subtype has been described. This subtype, termed the 5-HT1 binding site, is located in highest density in the choroid plexus (Pazos et at., 1984). Most interest, though, has been focused on defining the affinity and selectivity of 5-HT agonists and antagonists for the 5-HT1A and 5-HT1B Received for publication March 13, 1987. 1 This work was supported by U.S. Public Health Service Grante MH 29094, 14654 and GM 34781 and funds from the Veterans Administration. binding sites. At the 5-HT1B receptor, this characterization has been done using the nonselective radioligand [3H]5-HT (Sills et at., 1984a; Asarch et al., 1985). Interest in drugs interacting at 5-HT1A and 5-HT1B receptors has been stimulated by the fmding of specific responses mediated through each of these subtypes. For example, activation of 5-HT1A receptors inhibits forskolin stimulated adenylyl cyclase (De Vivo and Maayani, 1986). The inhibition of electricalor potassium-stimulated release of 5-HT is modulated by nerve terminal 5-HT autoreceptors (G#{246}thert, 1980; Cox and Ennis, 1982), which are thought to be 5-HT1B receptors (Engel et at., 1986; Raiteri et at., 1986). The 5-HT1A and 5-HT1B subtypes also mediate specific behaviors in rats. The 5-HT behavioral syndrome is a 5-HT1A receptor-mediated response (Lucki et at., 1984; Trickelbank et .at 1985) and the decrease in locomotion produced ABBREVIATIONS: 5-HT, 5-hydroxytryptamine (serotonin); TFMPP, 1-(m-t#{241}fluoromethylphenyl)-piperazine; m-CPP, m-chlorophenylpiperazine; CNS, central nervous system; DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin; ICYP, iodocyanopindolol; 5,7-DHT, 5,7-dihydroxytryptamine; SN, substantia nigra; CN-lMl, cyanoimipramine; LSD, d-lysergic acid diethylamide; DMT, N,N-dimethyltryptamine; 5-MeODMT, 5-methoxy-N,N-dimethyltryptamine; B , maximum number of binding sites. 144 at A PE T Jornals on July 7, 2017 jpet.asjournals.org D ow nladed from 5-HT1. Receptors and Drug Affinfty 145 1988 by piperazine derivatives like TFMPP and m-CPP may be a consequence of 5-HT1B receptor activation (Lucki and Frazer, 1985). In light of the finding that 5-HTIA and 5-HT1B receptors mediate quantifiable responses, the determination of drug affinity and, in particular, drug selectivity at these receptors has become important. In addition, considerable interest has been focused on the regulation of 5-HT receptors in the CNS, as regulatory effects on these receptors may be involved in the mechanism of action of antidepressants (Savage et at., 1980a; Peroutka and Snyder, 1980) and, perhaps, the behavioral tolerance that develops to hallucinogens (Trulson and Jacobs, 1979; Buckholtz et at., 1985). Conflicting reports exist regarding the effects of lesioning central serotonergic neurons on the density of subtypes of receptors for 5-HT (see Frazer et aL, 1988). These types of studies have been hampered by the lack of selective radioligands for subtypes of the 5-HT1 receptor. Early studies evaluated the 5-HT1 receptor using the agonist [3H]-5-HT. However, in the case of radiolabeled agonists like [3H1-5-HT, the ligand may label multiple states of the receptor (Sills et aL, 19Mb). If there are multiple affinity states, the high-affinity state must be eliminated by using a nucleotide such as GTP. This approach has disadvantages, as the addition of GTP reduces the amount of specifically bound radioligand which could make the analysis of competition curves difficult. Also, when this ligand has been used to study subtypes of the 5-HT1 receptor, it has been necessary to block its binding to one of the subtypes (Sills et at., 1984a; Peroutka, 1986) as 5HT binds with high affinity to both 5-HT1A and 5-HT1B receptors. In brain areas in which the subtype being measured is only a small percentage of the total number of receptors bound by [3H]-5-HT, this strategy is problematic. Consequently, investigators have searched for radioligands that are selective for subtypes of receptors for 5-HT. Radioligands selective for the 5-HT1A and 5-HT1B subtypes have been described. For example, the agonist [3HJDPAT, a compound which exhibits 1000-fold selectivity for the 5-HT1A vs. 5-HT1B site (Middlemiss and Fozard, 1983), has been used extensively to characterize the 5-HT1A receptor. More recently, [‘25I]ICYP, in the presence of excess isoproterenol to block its binding to beta adrenoceptors, has been reported to label 5HT18 receptors with high affinity and with selectivity (Hoyer et at., 1985a,b). The purpose of this study was 3-fold: 1) to optimize the assay conditions for the use of [IThI]ICYP; 2) to determine the affinity of 5-HT agonists and antagonists for the 5-HT1B subtype using this radioligand; 3) to use this ligand to determine the effects of destruction of serotonergic nerves on 5-HT1 receptors in specific areas ofthe CNS. Conditions were found in which [1251] ICYP bound to a single population of binding sites on which GTP had little effect. Also, regionally selective effects were produced on 5-HT1B receptors by the intraventricular administration of 5,7-DHT, namely the density of binding sites increased in the caudate-putamen but not in either the substantia nigra or cerebral cortex.