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LY334370 is a high affinity, selective agonist at the 5-HT(1F) receptor. On this basis, the tritiated compound was examined for its utility in autoradiography to localize the 5-HT(1F) receptor in rat and guinea pig brain regions. Specific 5-HT(1F) receptor binding in rat brain was found in layers 4-5 of all cortical regions examined, as well as olfactory bulb and tubercle, nucleus accumbens, caudate putamen, parafascicular nucleus of the thalamus, medial mammillary nucleus, the CA3 region of the hippocampus, subiculum, and several amygdaloid nuclei. In guinea pig brain, the [(3)H]LY334370 binding sites were found at highest density in claustrum, but also in a layer of the cortex, caudate putamen, nucleus accumbens, thalamus, and medial mammillary nucleus. Some species differences in the distribution of the 5-HT(1F) receptor were noted. Side by side comparison of rat brain autoradiography with [(3)H]LY334370 and [(3)H]sumatriptan showed labeling in the same brain regions. Preliminary binding studies in rhesus monkey and human brain sections showed [(3)H]LY334370 binding in cortical layers 4-5, subiculum (in the monkey), and the granule cell layer of the cerebellum. These findings suggest a discrete localization of the 5-HT(1F) receptor in the rat, guinea pig, monkey and human brain, and confirms the utility of [(3)H]LY334370 as a potential tool to explore further the localization and possible functions of the 5-HT(1F) receptor.

[(3)H]LY334370 was developed as a radioligand to study the characteristics of this compound's interaction with the 5-HT(1F) receptor. Monovalent or divalent cations did not enhance the binding of [(3)H]LY334370 to the cloned human 5-HT(1F) receptor. In the presence of MgCl(2), the time to reach equilibrium was approximately 2 h, while in its absence equilibrium was reached in less than 1 h. [(3)H]LY334370 had high affinity for the cloned human 5-HT(1F) receptor (K(d)=0.446 nM) and the 5-HT(1F) receptor in rat brain (K(d)=0.388 nM). The expression density of 5-HT(1F) receptors, as determined by binding to homogenates of cortical regions from rat, was low (B(max)=79.1 fmol/mg protein). There was a statistically significant correlation between the apparent pK(i) for inhibition of [(3)H]LY334370 binding and the pEC(50) for stimulation of [(35)S]GTPgammaS binding to homogenates of cells expressing the cloned human 5-HT(1F) receptor. In addition, there was a statistically significant correlation between the apparent pK(i) for inhibition of [(3)H]LY334370 binding to the cloned human 5-HT(1F) receptor and the pID(50) for inhibition of trigeminal nerve stimulated dural plasma protein extravasation in the guinea pig. The conclusion from these studies is that [(3)H]LY334370 is a high affinity radioligand which can be used for the study of the 5-HT(1F) receptor in rat brain or in cells transformed with the human 5-HT(1F) receptor.

Since the classical hallucinogens were initially reported to produce their behavioral effects via a 5-HT2 agonist mechanism (i.e., the 5-HT2 hypothesis of hallucinogen action), 5-HT2 receptors have been demonstrated to represent a family of receptors that consists of three distinct subpopulations: 5-HT2A, 5-HT2B, and 5-HT2C receptors. Today, there is greater support for 5-HT2A than for 5-HT2C receptor involvement in the behavioral effects evoked by these agents. However, with the recent discovery of 5-HT2B receptors, a new question arises: do classical hallucinogens bind at 5-HT2B receptors? In the present study we examined and compared the binding of 17 phenylisopropylamines at human 5-HT2A, 5-HT2B, and 5-HT2C receptors. Although there was a notable positive correlation (r>0.9) between the affinities of the agents at all three populations of 5-HT2 receptors, structural modification resulted only in small differences in 5-HT2B receptor affinity such that the range of affinities was only about 50-fold. As with 5-HT2A and 5-HT2C receptor affinity, there is a significant correlation (r>0.9, n=8) between 5-HT2B receptor affinity and human hallucinogenic potency. Nevertheless, given that 5-HT2A and 5-HT2A/2C antagonists - antagonists with low affinity for 5-HT2B receptors - have been previously shown to block the stimulus effects of phenylisopropylamine hallucinogens, it is likely that 5-HT2A receptors play a more prominent role than 5-HT2B and 5-HT2C receptors in mediating such effects despite the affinity of these agents for all three 5-HT2 receptor subpopulations.

In previous reports, [3H]5-HT has been used to characterize the pharmacology of the rat and human 5-HT2B receptors. 5-HT, the native agonist for the 5-HT2B receptor, has a limitation in its usefulness as a radioligand since it is difficult to study the agonist low-affinity state of a G protein-coupled receptor using an agonist radioligand. When using [3H]5-HT as a radioligand, rauwolscine was determined to have relatively high affinity for the human receptor (Ki human = 14.3+/-1.2 nM, compared to Ki rat = 35.8+/-3.8 nM). Since no known high affinity antagonist was available as a radioligand, these studies were performed to characterize [3H]rauwolscine as a radioligand for the cloned human 5-HT2B receptor expressed in AV12 cells. When [3H]rauwolscine was initially tested for its usefulness as a radioligand, complex competition curves were obtained. After testing several alpha2-adrenergic ligands, it was determined that there was a component of [3H]rauwolscine binding in the AV12 cell that was due to the presence of an endogenous alpha2-adrenergic receptor. The alpha2-adrenergic ligand efaroxan was found to block [3H]rauwolscine binding to the alpha2-adrenergic receptor without significantly affecting binding to the 5-HT2B receptor and was therefore included in all subsequent studies. In saturation studies at 37 degrees C, [3H]rauwolscine labeled a single population of binding sites, Kd = 3.75+/-0.23 nM. In simultaneous experiments using identical tissue samples, [3H]rauwolscine labeled 783+/-10 fmol of 5-HT2B receptors/mg of protein, as compared to 733+/-14 fmol of 5-HT2B receptors/mg of protein for [3H]5-HT binding. At 0 degrees C, where the conditions for [3H]5-HT binding should label mostly the agonist high affinity state of the human 5-HT2B receptor, [3H]rauwolscine (Bmax = 951+/-136 fmol/mg), again labeled significantly more receptors than [3H]5-HT (Bmax = 615+/-34 fmol/mg). The affinity of [3H]rauwolscine for the human 5-HT2B receptor at 0 degrees C did not change, Kd = 4.93+/-1.27 nM, while that for [3H]5-HT increased greatly (Kd at 37 degrees C = 7.76+/-1.06 nM; Kd at 0 degrees C = 0.0735+/-0.0081 nM). When using [3H]rauwolscine as the radioligand, competition curves for antagonist structures modeled to a single binding site, while agonist competition typically resulted in curves that best fit a two site binding model. In addition, many of the compounds with antagonist structures displayed higher affinity for the 5-HT2B receptor when [3H]rauwolscine was the radioligand. Typically, approximately 85% of [3H]rauwolscine binding was specific binding. These studies display the usefulness of [3H]rauwolscine as an antagonist radioligand for the cloned human 5-HT2B receptor. This should provide a good tool for the study of both the agonist high- and low-affinity states of the human cloned 5-HT2B receptor.