5-Methoxytryptoline, a Competitive Endocoid Acting at [3H]Imipramine Recognition Sites in Human Platelets

5-Methoxytryptoline potently inhibits [3H]imipramine binding to membranes from the cerebral cortex and platelets. Since 5-methoxytryptoline, which appears to occur endogenously with particularly high levels in the human pineal gland, also inhibits 5-hydroxytryptamine (5-HT, serotonin) uptake, it should be considered as a putative endogenous ligand modulating 5-HT transport. As the 5-HT transporter complex comprises the imipramine and the substrate recognition sites, which interact allosterically, it was essential to define the mechanism of inhibition of [3H]imipramine binding by 5-methoxytryptoline. Human platelets show an active and saturable uptake of 5-HT and tryptamine. The uptake of both substrates appears to be mediated by the same carrier and it is inhibited by 5-methoxytryptoline at submicromolar concentrations. 5-HT and tryptamine inhibit [3H]imipramine binding in human platelets with a Hill slope for inhibition close to unity and IC50 values of 3,265 and 3,475 nM, respectively. This inhibition is, however, not competitive because both 5-HT and tryptamine significantly decrease the rate of [3H]imipramine-receptor dissociation. Although 5-methoxytryptoline potently inhibits [3H]imipramine binding (IC50 = 44 nM) in human platelets with a Hill slope of unity, it does not affect the receptor-ligand dissociation rate of [3H]imipramine even at concentrations up to 100 microM. The present experiments show that 5-methoxytryptoline, in spite of its chemical similarity to the indoleamine transporter substrates, interacts with the imipramine receptor through a mechanism of competitive inhibition. This conclusion is supported by a selective effect of 5-methoxytryptoline on the Kd of [3H]imipramine binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between levels and uptake of serotonin and high affinity [3H]imipramine recognition sites in the rat brain

High affinity [3H]imipramine binding, endogenous levels of serotonin and noradrenaline, and serotonin uptake were determined in brain regions of rats with selective destruction of serotonergic neurons by 5,7-dihydroxytryptamine (5,7-DHT), of adrenergic neurons by 6-hydroxydopamine (6-OHDA), and of rats treated with reserpine. Neonatal treatment with 5,7-DHT resulted in a significant decrease of both serotonin levels and density (Bmax) of high affinity [3H]imipramine binding sites in the hippocampus. In contrast, an elevation of serotonin levels and an increase in Bmax of [3H]imipramine binding were noted in the pons--medulla region. No changes were observed in the noradrenaline content in either of these regions. Intracerebral 6-OHDA lesion produced a drastic suppression of noradrenaline levels in cerebral cortex but failed to alter the binding affinity (KD) or density (Bmax) of [3H]imipramine recognition sites. A single injection of reserpine (2.5 mg/kg) resulted in marked depletion of both serotonin (by 57%) and noradrenaline (by 86%) content and serotonin uptake (by 87%) in the cerebral cortex but had no significant influence of the parameters of high affinity [3H]imipramine binding in this brain region. The results suggest that high affinity [3H]imipramine binding in the brain is directly related to the integrity of serotonergic neurons but not to the magnitude of the uptake or the endogenous levels of the transmitter, and is not affected by damage to noradrenergic neurons or by low levels of noradrenaline.     

"Specific" Binding of [3H]Imipramine to Protease-Sensitive and Protease-Resistant Sites

A number of 5-hydroxytryptamine (5-HT) uptake inhibitors have been shown to displace the binding of [3H]imipramine to rat cortical membranes in a complex manner with Hill slopes less than unity. Norzimeldine displaced the binding of [3H]imipramine in a biphasic manner with IC50 values for the two components of about 30 nM and 30 microM. This latter site alone was found in tissues that had been treated with a protease. Binding to both of these sites was displaced by 10 microM desipramine. The protease-sensitive [3H]imipramine binding sites were found to be saturable, high-affinity binding sites with a KD of 8 nM. The number of these sites varied between brain regions and was positively correlated with the regional distribution of [14C]5-HT but not [3H]noradrenaline uptake. This was not the case however for the protease-resistant but desipramine-displaceable binding sites. Since most previous [3H]imipramine binding studies have been performed with high concentrations of desipramine (10 microM) to define "specific binding," these data would suggest that either protease-sensitivity or displacability by 1 microM norzimeldine would give more reliable estimates of the specific binding.     

Sodium-dependent [3H]imipramine binding in rat hippocampus and its relationship to serotonin uptake

The relationship of [3H]imipramine recognition sites and serotonergic function was investigated by simultaneously determining the desipramine-defined and sodium-dependent components of [3H]imipramine binding and the serotonin levels and uptake in hippocampus of rats without and with selective lesion of serotonergic neurons with 5,7-dihydroxytryptamine. In control rats, the desipramine-defined [3H]imipramine binding to hippocampal membranes showed a high affinity (Kd = 2 nM) and low affinity (Kd = 31 nM) component. In contrast, the Scatchard analysis of sodium-dependent binding revealed a single class of sites of high affinity (Kd = 1.5 nM). Displacement of sodium-dependent [3H]imipramine binding by cold imipramine resulted in a steep curve best fitted to a one-site model. Sodium-dependent binding of [3H]imipramine at 4 nM concentration represented only about 38% of desipramine-defined binding. 5,7-Dihydroxytryptamine treatment resulted in marked reduction of hippocampal serotonin concentration and uptake without any changes in norepinephrine levels. Virtually only the low affinity component of desipramine-defined [3H]imipramine binding was detected by Scatchard analysis in 5,7-dihydroxytryptamine lesioned rats. The desipramine-defined "specific" [3H]imipramine binding in hippocampi of lesioned rats was decreased by 46%, whereas the sodium-dependent binding was only 18% of that seen in controls. Desipramine-defined specific binding in absence of sodium was not altered by lesion to serotonergic neurons. The results suggest that desipramine-defined specific [3H]imipramine binding may not be appropriate for studying the role of imipramine sites in relation to serotonin neuronal uptake and that determination of sodium-dependent binding components of both [3H]imipramine binding and serotonin uptake should be used in future studies.     

Elevated Density of [3H]Imipramine Binding in Aged Human Brain

Aging was associated with an increase in the density of specific binding sites for [3H]imipramine in postmortem specimens of human hypothalamus, frontal cortex, and parietal cortex. In general, [3H]imipramine binding was not affected by factors considered difficult to control in postmortem studies, i.e., time from death to autopsy and cause of death. The in vitro regulation of [3H]imipramine binding by sodium was impaired with age in hypothalamic homogenates. In vitro regulation of [3H]imipramine binding by chloride was intact. Determination of the concentrations of 5-hydroxytryptamine (serotonin) and 5-hydroxyindoleacetic acid in hypothalamus and frontal cortex indicated no apparent age-related changes in indole metabolism. The age-related increase in brain [3H]imipramine binding and impairment in the in vitro regulation of binding by ions are similar to changes observed previously in aged mouse brain. The increase in brain antidepressant binding sites is discussed in relationship to other indices of brain serotonergic function in aging and to the relationship of [3H]imipramine binding and depression.     

Subdivision of Mouse Brain [3H]Imipramine Binding Based on Ion Dependence and Serotonin Sensitivity

The specific binding of [3H]imipramine to mouse brain membranes in an assay containing 120 mM NaCl and 5 mM KCl was similar in regional distribution and pharmacological specificity to that reported previously in rat and human brain. However, the absence of ions decreased the density of the specific binding of [3H]imipramine and did not affect the equilibrium dissociation constant. Sodium was the only cation, and halides were the only anions tested that enhanced the specific binding of [3H]imipramine. Chloride did not increase the density of binding in the absence of sodium. The ion-sensitive binding of [3H]imipramine was regionally dependent and was highly correlated with the uptake of 5-hydroxytryptamine (5-HT, serotonin) into synaptosomes from brain regions. 5-HT did not inhibit the binding of [3H]imipramine in the absence of ions. Antidepressants inhibited binding in the absence and presence of ions, but in the presence of ions inhibition curves were shifted to the left and the apparent complexity of inhibition was increased. Quantitative analysis of the inhibition of [3H]imipramine binding by antidepressants conducted in the presence of ions was consistent with two binding sites. Lesion of the serotonergic input to the cerebral cortex by 5,7-dihydroxytryptamine suggested that both the 5-HT-sensitive and ion-sensitive binding of [3H]imipramine were associated with serotonergic nerve terminals. [3H]Imipramine binding displaced by desipramine, but insensitive to 5-HT and ions, was not affected by the lesion. Thus, the binding of [3H]imipramine that is displaced by desipramine, the most common assay for [3H]imipramine binding, includes a component that is not associated with brain serotonergic nerve terminals and 5-HT uptake, and, in addition, a separable component that is highly correlated with serotonergic function. These data have important implications for studies of serotonergic neurons and for the interpretation of imipramine binding data.     

The Effect of Endogenous Modulator Endobain E on NMDA Receptor Is Interfered by Zn22+ but Is Independent of Modulation by Spermidine

A brain endogenous factor, termed endobain E, allosterically decreases [3H]dizocilpine binding to NMDA receptor. Such effect depends on receptor activation by the coagonists glutamate and glycine and is interfered by channel blockers, suggesting its interaction with the inner surface of the associated channel. To further analyze endobain E effect on NMDA receptor, in the current study competitive [3H]dizocilpine binding assays to brain membranes were performed with Zn2+ to block the associated channel, as well as with spermidine (SPD), which exerts positive allosteric modulation of NMDA receptor. Partially or nonadditive effects on [3H]dizocilpine binding were recorded, respectively, in the presence of endobain E at a concentration that inhibits binding 25% plus IC25 Zn2+ or endobain E at a concentration that inhibits binding 50% plus IC50 Zn2+. With an endobain E concentration that decreases 25% ligand binding, SPD potentiated binding over a wide concentration range but failed to modify endobain E effect. Similarly, [3H]dizocilpine binding reduction over a wide endobain E concentration range remained unaltered by high SPD concentrations. Additive effects were observed with endobain E at a concentration that decreases binding 25% plus IC25 SPD site antagonists arcaine or ifenprodil. Zn2+ experiments indicated that endobain E effect is interfered by channel blockade produced by this ion. Although endobain E effect is dependent on NMDA receptor activation by glutamate and glycine, it proves independent of the positive modulation exerted by SPD. Thus the endogenous modulator seems not to interact at NMDA receptor polyamine site, favoring the hypothesis that endobain E binds inside the associated channel.     

Assessment of rat brain alpha1-adrenoceptor binding and activation of inositol phospholipid turnover following chronic imipramine treatment

Chronic (21 days) treatment of rats with imipramine (10 mg/kg) did not change the density or affinity of alpha₁-adrenoceptors as measured by the specific binding of [³H]prazosin in rat cortical membranes, but produced the expected significant decrease in the density of beta-adrenoceptors labeled by [¹²⁵I]iodocyanopindolol. The functional status of brain alpha₁-adrenoceptors was also assessed by measuring the noradrenaline (NA)-induced accumulation of [³H]inositol 1-phosphate (IP₁) in brain slices from these animals. No apparent change was observed in the concentration-response relationship between NA and [³H]IP₁ accumulation in rat cerebral cortex after chronic treatment with imipramine. At concentrations higher than 1 M in vitro, imipramine and its metabolite, desipramine, produced a concentration-dependent decrease in the [³H]IP₁ accumulation elicited by NA. This inhibitory effect is likely mediated by direct blockade of alpha₁-adrenoceptors by these drugs. As the endogenous drug concentration would not reach 1 M in our preparation, the lack of changes in alpha₁-adrenoceptor response following chronic imipramine treatment are not likely attributable to residual imipramine or desipramine retained in the tissues. In conclusion, the above findings do not support previous suggestions that brain alpha₁-adrenoceptors are upregulated following chronic imipramine administration.     

[3H]Imipramine Labels Sites on Brain Astroglial Cells Not Related to Serotonin Uptake

Brain astroglial cells, whether from a bulk isolated preparation or in culture, have been shown to take up serotonin actively. [3H]imipramine has been proposed as a specific label for serotonin uptake sites in brain. We therefore studied the binding of [3H]imipramine to C6 astroglial cells in culture to determine if some of the binding of this radioligand in brain homogenates is actually to serotonin transporting sites on glia. [3H]Imipramine binds saturably (Bmax = 202 fmol/mg protein) and with high affinity (KD = 1.72 nM) to C6 cells. This binding is competitively inhibited by other tricyclic antidepressants. The C6 cells actively transport [3H]serotonin with a Km of 2 microM and a Vmax of 1080 fmol/10(6) cells/min. However, the pharmacological profile for inhibition of serotonin uptake does not correlate with the pharmacological profile for inhibition of [3H]imipramine binding. These results suggest that the binding of [3H]imipramine to astroglial cells is not related to their capacity for active uptake of serotonin. Further, in brain homogenates, some of the binding of [3H]imipramine may not be to neuronal uptake sites but rather may be to sites on astroglial cells.     

Demonstration of specific "high affinity" binding sites for [3H] imipramine on human platelets

High affinity and saturable binding sites for [³H] imipramine have been demonstrated on human platelet membranes. These binding sites appear to be specific for tricyclic antidepressants and their pharmacologically-active metabolites. In contrast, inactive tricyclic compounds such as the parent iminodibenzyl and iminostilbenes do not inhibit [³H] imipramine binding. The binding of [³H] imipramine to human platelets is of high affinity $\text{(}\text{Kd}\text{? 1.4}\text{nM}\text{)}$, saturable $\text{(}\text{Bmax}\text{? 625}\text{fmols/mg prot}\text{)}$, and sensitive to proteolytic degradation. The effects of various drugs and neurotransmitter agonists and antagonists suggests that these binding sites are pharmacologically distinct from the previously reported binding of tricyclic antidepressants to alpha-adrenergic, muscarinic-cholinergic, and histaminergic receptors. The binding characteristics of [³H] imipramine to platelets is similar to that in rat and human brain and may thus serve as a useful model in elucidating the pharmacological and physiological significance of these binding sites.     

Does high affinity [3H] imipramine binding label serotonin reuptake sites in brain and platelet?

Recent studies have demonstrated the presence of high affinity binding sites for [³H] imipramine in membrane preparations derived from rat brain, human platelet, and human brain. Although initial reports concluded that there was no relationship between these binding sites and the reuptake sites for biogenic amines, subsequent studies in our laboratory suggested a close relationship between the high affinity imipramine binding site and the serotonin uptake or transport site (cf. ref. 9). To further establish whether these binding sites are associated with either platelet or neuronal uptake of serotonin, the relative potencies of a series of tricyclic antidepressants in inhibiting [³H] imipramine binding and serotonin uptake were determined under identical assay conditions. A close correlation between inhibition of serotonin uptake and [³H] imipramine binding was observed (r = 0.99, p < 0.001). In addition, electrolytic lesions of the midbrain raphe produced a decrease in [³H] imipramine binding in hypothalamic synaptosomes that paralleled the decrease in [³H] serotonin uptake. Finally incubation of synaptosomal membranes with 2,8-dinitroimipramine, an irreversible inhibitor of [³H] imipramine binding, produced a dose-dependent decrease in serotonin uptake, without altering the uptake of nonrepinephrine or dopamine. Taken together our results strongly suggest that high affinity binding of [³]] imipramine selectively labels serotonin uptake sites in brain and platelet.     

Binding of [3H]Imipramine to Mouse Cerebrocortical Membranes and to Glass Fiber Filters

It was suggested in a recent report by Phillips et al. [J. Neurochem. 43, 479-486 (1984)] that the low-affinity binding of [3H]imipramine in the mouse cerebral cortex could in fact represent binding of [3H]imipramine to the GF/B glass fiber filters used to terminate the assays. The present study demonstrates that this is not the case and advances two lines of evidence: (a) For saturation analysis, mouse cerebrocortical membranes were incubated with [3H]imipramine concentrations between 0.8 nM and 3.6 microM, and parallel incubations were carried out with buffer replacing the brain membranes. The same low-affinity component, in addition to the high-affinity component, was present in the binding of [3H]imipramine to brain membranes plus GF/B filters (uncorrected data), and in that to brain membranes alone (corrected data). (b) Dissociation experiments, in which filter binding is equal for all samples and dissociation time is the only variable, clearly indicated the nonhomogeneity of [3H]imipramine binding. Our results, however, do show that binding to recently purchased GF/B filters is not a negligible phenomenon in saturation experiments. Relatively lower binding was found to GF/C, GF/F, Gelman A/E, and Reeves Angel 934 AH filters; pretreatment of GF/B filters with polyethyleneimine (PEI) reduced binding to a greater extent in the single manifold than in the cell harvester.     

2-Nitroimipramine: A selective irreversible inhibitor of [3H] serotonin uptake and [3H] imipramine binding in platelets

The effect(s) of a new imipramine analogue, 2-nitroimipramine, on high affinity [³H] imipramine binding and [³H] serotonin uptake in human platelets were studied. 2-Nitroimipramine was found not only to be a very potent inhibitor of [³H] imipramine binding and [³H] serotonin uptake but was found to irreversibly inhibit binding and uptake simultaneously. This finding supports previous observations from our laboratory and others that high affinity imipramine binding labels serotonin uptake or transport sites. 2-Nitroimipramine should prove an important tool for subsequent studies of the molecular mechanism(s) involved in the transport of serotonin and the binding of imipramine to platelet and brain membranes.     

Demonstration of specific high affinity binding sites for [3H] imipramine in human brain

High affinity binding sites (Kd = 1.7 nM) for [³H] imipramine have been characterized in membranes prepared from human brain. The binding of [³H] imipramine was found to be saturable, reversible, and inhibited by pharmacologically active tricyclic antidepressants. Other psychoactive compounds as well as most neurotransmitter substances were ineffective in inhibiting [³H] imipramine binding at concentrations up to 10 μM. The hypothalamus was found to contain a relatively high density of these binding sites and is enriched approximately 4-fold when compared to cerebral and cerebellar cortex. A very good correlation (r = 0.97) p < 0.001 was found between the abilities of a series of clinically active tricyclic antidepressants in displacing specifically bound [³H] imipramine from human brain and platelet membranes, suggesting that the binding sites from these two tissues are very similar.     

Binding of imipramine and cocaine to a model lipid membrane

[³H]Imipramine and [³H]cocaine were concentrated at membranes of liposomes prepared from phosphatidylcholine, cholesterol, and dicetylphosphate. This binding has an apparent dissociation constant in the micromolar range and a density close to 2 pmol/g of phosphatidylcholine. The potencies of various drugs in inhibiting the binding ot liposomes correlated only weakly with those in inhibiting the high-affinity binding of [³H]imipramine and [³H]cocaine to brain membranes. However, there was a highly significant correlation between the potencies of drugs in inhibiting binding to liposomes and their lipophilic character, indicating the involvement of hydrophobic bonding. Although the amounts of phosphatidylcholine and cholesterol in brain preparations in assays for high-affinity binding to brain membranes were in the same range as those used in our assays with liposomes, the inhibition of the high-affinity binding to brain membranes was only weakly dependent upon the lipophilicity of the inhibiting drug. These results indicate that lipophilicity is but one of the factors in the complex binding interactions between lipophilic substances and integral brain membranes. In addition, the results are in agreement with the suggestion that phosphatidylcholine and cholesterol are not the primary sites of high-affinity binding [³H]imipramine and [³H]cocaine to brain membranes, although it cannot be ruled out that these lipids have different properties in natural biological membranes and in artificial liposome membranes.     

5-Hydroxytryptamine Uptake and Imipramine Binding Sites in Neurotumor NCB-20 Cells

NCB-20 cells (neuroblastoma X fetal Chinese hamster brain hybrids) are equipped with a [3H]5-hydroxytryptamine [( 3H]5-HT) uptake system and [3H]imipramine recognition sites. Approximately 80% of the radioactivity taken up by cells incubated with [3H]5-HT was identified with 5-HT. [3H]5-HT uptake was temperature-dependent, partially sodium-dependent, saturable (Km = 7.3 +/- 0.6 microM; Vmax = 2.0 +/- 0.6 pmol/min/mg), and inhibited by clomipramine, imipramine, fluoxetine, and desipramine, but not by iprindole, mianserin, or opipramol. Lineweaver-Burk plots showed a competitive type of inhibition by imipramine and fluoxetine. [3H]5-HT uptake was not inhibited by nisoxetine or benztropine. [3H]Imipramine binding sites had a KD of 12 +/- 2 nM and a Bmax of 22 +/- 7 pmol/mg protein. The binding was sodium-sensitive although to a lesser extent than that found with brain membranes. Imipramine binding was displaced by tricyclic antidepressants with the following order of potency: clomipramine greater than imipramine greater than fluoxetine greater than desipramine much greater than iprindole = mianserin greater than opipramol. These results suggest that imipramine binding sites are present together with the 5-HT uptake sites in NCB-20 cells and that these sites interact functionally but are different biochemically.     

Complex Inhibition of [3H]Imipramine Binding by Serotonin and Nontricyclic Serotonin Uptake Blockers

Tricyclic antidepressant drugs inhibit [3H]imipramine binding to the rat brain cortex in a competitive manner, giving linear Hofstee plots and Hill coefficients of approximately 1.0. Serotonin, the only neurotransmitter to inhibit [3H]imipramine binding, does so in a complex manner, exhibiting a Hill coefficient of 0.40-0.50. Nontricyclic inhibitors of serotonin uptake such as fluoxetine, paroxetine, norzimelidine, and citalopram inhibit [3H]imipramine binding in the same complex manner as serotonin. These results are interpreted as suggesting that [3H]imipramine binds to a site associated with the serotonin uptake system but different from either the substrate recognition site for serotonin or the site of action of the nontricyclic inhibitors of neuronal uptake of serotonin.     

Characterization of [3H]Paroxetine Binding in Rat Brain

The binding of the 5-hydroxytryptamine (5-HT, serotonin) uptake inhibitor [3H]paroxetine to rat cortical homogenates has been characterized. The effect of tissue concentration was examined and, with 0.75 mg wet weight tissue/ml in a total volume of 1,600 microliter, the binding was optimized with an apparent dissociation constant (KD) of 0.03-0.05 nM. Competition experiments with 5-HT, citalopram, norzimeldine, and desipramine revealed a high (90%) proportion of displaceable binding that fitted a single-site binding model. Fluoxetine and imipramine revealed, in addition to a high-affinity (nanomolar) site, also a low-affinity (micromolar) site representing approximately 10% of the displaceable binding. The specificity of the [3H]paroxetine binding was emphasized by the fact that 5-HT was the only active neurotransmitter bound and that the serotonin S1 and S2 antagonist methysergide was without effect on the binding. Both 5-HT- and fluoxetine-sensitive [3H]paroxetine binding was completely abolished after protease treatment, suggesting that the binding site is of protein nature. Saturation studies with 5-HT (100 microM) sensitive [3H]paroxetine binding were also consistent with a single-site binding model, and the binding was competitively inhibited by 5-HT and imipramine. The number of binding sites (Bmax) for 5-HT-sensitive [3H]paroxetine and [3H]imipramine binding was the same, indicating that the radioligands bind to the same sites. Lesion experiments with p-chloroamphetamine resulted in a binding in frontal and parietal cortices becoming undetectable and a greater than 60% reduction in the striatum and hypothalamus, indicating a selective localization on 5-HT terminals. Together these findings suggest that [3H]paroxetine specifically and selectively labels the substrate recognition site for 5-HT uptake in rat brain.     

Effects of thyroid status on the characteristics of alpha 1-, alpha 2-, beta, imipramine and GABA receptors in the rat brain.

The effects of a chronic treatment with L-triiodothyronine (T3; 100 mg/rat/day s.c. for 7 days) or with propylthiouracil (PTU; 50 mg/rat/day for 35 days by stomach tube) on the characteristics of alpha 1, alpha 2, beta, imipramine and GABA binding sites in different brain areas of the adult rat have been studied. T3-treatment caused an increase in the number of [3H]dihydroalprenolol and a decrease in the number of [3H]muscimol binding sites in the cerebral cortex. PTU-treatment caused a decrease in the number of [3H]prazosin, [3H]yohimbine and [3H]dihydroalprenolol binding sites in the cerebral cortex, while the number of [3H]imipramine binding sites was reduced in the cerebral cortex and hypothalamus, and increased in the hippocampus. Affinity constants were never modified. Concurrent experiments showed that the "in vitro" addition of T3 and PTU did not influence the binding of any of the ligands employed to control rat brain membranes. The present data further support the view that neurotransmission in the CNS is influenced by the thyroid status.     

Evidence for an endogenous peptide ligand for the phencyclidine receptor

Porcine brain contained an active factor that competed with [3H]-phencyclidine (PCP) for binding to rat brain membranes. On reverse phase high pressure liquid chromatography, the active material eluted between 38-42% acetonitrile. Gel filtration chromatography of the factor predicted a molecular weight of approximately 3000 daltons. The endogenous substance appeared to be selective for PCP receptors as it did not interact with either benzodiazepine, neurotensin, nor with mu, delta, or kappa opioid receptors. The active material showed a heterogenous distribution in brain, with highest concentrations found in hippocampus and cortex. It is likely to be a small peptide since various proteases eliminated or markedly reduced the potency of the compound in a [3H]-PCP binding assay. The material also possessed PCP-like activity in two bioassays. Like PCP, it induced contralateral rotational behavior after unilateral intranigral injection and depressed spontaneous cell activity after iontophoretic micropressure application in hippocampus and cerebral cortex. Thus, this small peptide is likely to be an endogenous ligand for the PCP receptor.