Pii: S0304-3940(00)01614-1

Derivatives of phenylethylamine and tryptamine share structural features with the non-competitive N-methyl-d-aspartate (NMDA) receptor blockers phencyclidine, ketamine, and MK-801. Tryptamine and phenylethylamine inhibited the speci®c binding of [H]MK-801 to rat hippocampal membranes with IC50`s 190 and 905 mM, respectively. The corresponding amino acids phenylalanine and tryptophan were inactive, their methyl esters, however, were slightly more potent than the amines. The methyl ester of the naturally occurring l-tryptophan was 12 times more potent than the methyl ester of the d-isomer, whereas the corresponding isomers derived from phenylalanine exhibited no stereoselectivity. The potency of tryptamine was increased by substitutions as, e.g. in 5-methyltryptamine (IC50 12 mM) and tryptophan octylester (IC50 5.2 mM). Compounds formed in vivo from l-tryptophan and a lipophilic counterpart may function as endogenous non-competitive NMDA receptor blockers. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tryptamine; N-methyl-d-aspartate receptor; Rat hippocampus; [H]MK-801; Binding site; Endogenous ligand The ion channel associated with the N-methyl-d-aspartate receptor (NMDA)-receptor complex is the target of dissociative anesthetics like ketamine and phencyclidine (PCP). In 1987, Zukin et al. [12], partially puri®ed a fraction from bovine hippocampal extracts mimicking the actions of PCP on the binding of [H]TCP (the thieno-analog of PCP) and on NMDA-stimulated release of acetylcholine and dopamine. Already 3 years earlier, at a time when PCP still was not known to act via the NMDA receptor complex, Quirion et al. [9] had isolated from porcine brain a peptide with an estimated molecular weight of 3000 Da whose binding and physiological properties were similar to PCP and coined the term a-Endopsychosin. In 1994, Porter and Greenamyre [8] reported about an endogenous inhibitor of [H]MK-801 binding that could be eliminated from rat brain tissue sections by a prewash; the washing procedure was more ef®cient in the presence of the co-agonists glutamate and glycine, i.e. when the ion channel was in its open state. And in 1998, combinatorial chemistry led to the discovery of several hexapeptides rich in tryptophan and arginine residues, potently inhibiting glutamate-evoked currents at NMDA receptors expressed in Xenopus oocytes [3]. Whether any of these latter peptides (or compounds structurally related to them) were responsible for the endogenous activities observed earlier is unknown. In search for simple endogenous compounds ful®lling obvious requirements for blocking an ion channel permeable to cations (positive charge and lipophilicity), physiological aromatic amines derived from phenylethylamine and indolethylamine (tryptamine) were screened for their potency as inhibitors of [H]MK-801 binding. Parts of the present results have been published in abstract form [2]. The CA1/dentate gyrus part of rat hippocampi was dissected from unfrozen rat brains (male Wistar rats, age 4±7 months) and homogenized in cold 50 mM Tris±acetate (pH 7.0) with a glass/te ̄on Potter-type homogenizer. Whole cell membranes were obtained by centrifugation (10 min 35 000 £ g) and washed four times, including a 10 min treatment with 0.02% Triton X-100 in the 2nd washing step (378C water bath). Aliquots of membrane suspension were kept at 2808C for several years without loss of binding. [H]MK-801 (NEN, 5 nM, 23.9 Ci/mmol) was bound to thawn membranes (equivalent to 1 mg original tissue/vial) in 50 mM Tris±acetate (pH 7.0), in the presence of 10 mM glutamate and glycine (2 h, 238C water bath). All determiNeuroscience Letters 296 (2000) 29±32 0304-3940/00/$ see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(00)01614-1 www.elsevier.com/locate/neulet * Tel.: 143-1-4277-62892; fax: 143-1-4277-62899. E-mail address: [email protected] (M.L. Berger). nations were performed in duplicates or triplicates. For nonspeci®c binding (NB) glutamate and glycine were replaced by their antagonists D-2-amino-5-phosphono valeric acid (100 mM) and 5,7-dichlorokynurenic acid (10 mM; both Tocris Cookson Ltd). Inhibition of speci®c [H]MK-801 binding by test compounds was computer ®tted to the formula Bound ligand ˆBoICH 50 =…ICH 50 1…inhibitor†H †1 NB where Bo is the amount of ligand bound speci®cally in the absence of inhibitor and nH is the Hill coef®cient. Total binding (in the absence of inhibitor) amounted to 1500±2500 dpm/vial, NB to 150±300 dpm/vial. In experiments conducted in parallel under the same conditions, KD values for the radioligand between 10 and 15 nM were obtained (not shown). Results in Tables 1±4 are given as means ^ SD (n, number of experiments; if only two experiments were performed, both values are indicated). 2-Methyl-5-OH-tryptamine (2-methyl-serotonin) was obtained from Tocris Cookson Ltd. 5-Fluoro-a-methyltryptamine was a kind gift from M. BoÈs, Hoffmann-La Roche Ltd., Basel. 1-Methyltryptamine, 5-methyltryptamine, and 6-methyltryptamine were provided by Research Biochemicals International (RBI) as part of the Chemical Synthesis Program of the NIMH, Contract N01MH30003. All other test compounds were obtained from Aldrich or from Sigma. Tryptamine inhibited [H]MK-801 binding more potently (IC50 190 mM) than phenylethylamine (IC50 905 mM, Table 1, Fig. 1). N-methylation, hydroxylation, methoxylation, and ring formation had only a minor in ̄uence; RS-synephrine, RS-octopamine, RS-phenyl-ethanolamine, R-adrenaline, and R-noradrenaline produced less than 50% inhibition at 3 mM (not shown). a-Substitution was tolerated in both structural leads (Table 2). Remarkably, in a-carboxy-substituted tryptamine the S-con®guration (as in natural l-tryptophan) was preferred to the R-con®guraM.L. Berger / Neuroscience Letters 296 (2000) 29±32 30 Table 1 Inhibition of [H]MK-801 binding by phenylethyland indolethylamines: in ̄uence of N-methylation, hydroxylation, methoxylation, and ring formation